Use of Essential Oils for the Control of Varroa Jacobsoni Oud. in Honey Bee Colonies Anton Imdorf, Stefan Bogdanov, Rubén Ibáñez Ochoa, Nicholas W

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Use of essential oils for the control of Varroa jacobsoni Oud. in honey bee colonies Anton Imdorf, Stefan Bogdanov, Rubén Ibáñez Ochoa, Nicholas W. Calderone

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Anton Imdorf, Stefan Bogdanov, Rubén Ibáñez Ochoa, Nicholas W. Calderone. Use of essential oils for the control of Varroa jacobsoni Oud. in honey bee colonies. Apidologie, Springer Verlag, 1999, 30 (2-3), pp.209-228. hal-00891579

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Use of essential oils for the control of Varroa jacobsoni Oud. in honey bee colonies

Anton Imdorf Stefan Bogdanova Rubén Ibáñez Ochoaa Nicholas W. Calderone

a Forschungsanstalt für Milchwirtschaft, Sektion Bienen, Liebefeld, CH-3003 Bern, Switzerland b Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853, USA

(Received 3 September 1998; accepted 8 February 1999)

Abstract - Essential oils and essential oil components offer an attractive alternative to synthetic acaricides for the control of Varroa jacobsoni. They are generally inexpensive and most pose few health risks. Terpenes (mainly monoterpenes) are the main components of essential oils, comprising about 90 % of the total. More than 150 essential oils and components of essential oils have been evaluated in laboratory screening tests. Very few of them, however, have proven successful when tested in field trials. Thymol and thymol blended with essential oils or essential oil components offer a promis- ing exception. Mite mortality obtained with these formulations typically exceeds 90 % and often approaches 100 %. In addition, residues in honey are low, even after long-term treatments. The exact conditions under which these formulations will yield reliable and effective control, however, have only been determined for certain European regions. Based on the available studies, relying solely on a sin- gle treatment with an essential oil or essential oil component is generally not sufficient to maintain mite populations below the economic injury level. Therefore, efforts are necessary to optimize the use of these substances and to incorporate them, along with other measures for limiting mite populations, into an integrated pest management strategy for control of Varroa jacobsoni. © Inra/DIB/AGIB/ Elsevier, Paris

Apis mellifera / Varroa jacobsoni / essential oil / screening / treatment / residue

1. INTRODUCTION that had been introduced into the home range The association of Varroa jacobsoni of A. cerana, the mite’s original host. Oudemans (Acari: Varroidae) with the west- V. jacobsoni has subsequently established ern honey bee, Apis mellifera L., reportedly a nearly world-wide distribution with respect originated in the 1950s [23, 45, 70, 87] when to its new host [36, 70], with Australia being the mites transferred to A. mellifera colonies the only continent free of infestation.

* Correspondence and reprints E-mail: [email protected] V. jacobsoni and A. cerana maintain a stable 2. PROPERTIES OF ESSENTIAL OILS relationship. However, mortality in colonies of A. mellifera in temperate regions approa- ’Essential oils’ is the generic term for ches 100 % [24, 25]. Colonies in temperate liquid, highly volatile plant compounds, must be treated once or twice a regions year characterized by an intensive, characteris- to maintain mite below eco- populations tic odor. They are present in almost all plant nomic levels. the last 10 injury During years, species, but only plants containing more pyrethroids have been the primary source than 0.1 % oil can be called essential oils. of insecticides used to control V. jacobsoni. Essential oils are plant products found in mites in of have Recently, parts Europe the flowers, fruits, seed, leaves, roots or resistance to two flu- developed pyrethroids, wood and they can be present in all parts of valinate and flumethrin 21, 30, 32, [4, 27, a plant (e.g. Pinaceae) or only in specific and to couma- 63, 66, 74, 75, 89, 92, 96] parts (e.g. flowers of roses). Essential oils an phos, organophosphate [61]. Fluvalinate serve various functions in plants. They may resistance has also been documented in the act as attractants for insect pollinators; or, US The use of [5]. widespread synthetic they can be repellent, providing protection lipophilic acaricides has lead to the accu- against phytophagous insects. Many also mulation of residues in beeswax, propolis exhibit fungicidal and bactericidal activity, and to a much lesser degree, in honey (see protecting plants from microorganisms. [9] for further references). The essential oil of each The of acaricide resistance composition development plant species tends to be unique. However, in V. jacobsoni populations and the spectre some species have varieties, the so-called of the contamination of hive products pro- chemotypes, with variable essential oil com- vide considerable incentive to new develop for treatment that minimize the positions. Thyme (Thymus vulgaris), strategies poten- has at least seven tial for the of resistance example, chemotypes. rapid development One consists of the so-called and the accumulation of residues. Since group ’strong chemotypes’ that contain higher concentra- V. jacobsoni was introduced into Europe, tions of the phenols thymol and carvacrol, intensive efforts have been made to develop while a second group, the ’mild chemo- alternative chemical control measures based includes varieties with on formic, lactic and oxalic acids, combined types’, higher amounts of alcohols such as geraniol, with biotechnical measures (see [48] for fur- linalool and The chemical com- ther references). thujanol. position of an essential oil often depends It is well known that many essential oils upon cultivation and climatic conditions. and their components exhibit acaricidal The specific extraction process used to pro- activity. Before V. jacobsoni was a world- duce an essential oil also influences its com- wide pest, Angelloz [3] and Vecchi and position. Vapor distillation, cold pressing Giordani [93] tested different components of and chemical extractions produce oils of essential oils for their activity against Acara- varying composition. The lack of constancy pis woodi. Methyl salicylate and menthol in the chemical composition of essential oils proved to be toxic to the tracheal mite. In undoubtedly contributes to the variation in North America, where A. woodi has become results obtained by different researchers (see an important bee parasite, menthol is widely section 4). used in control efforts (see [ 16] for further The main chemical of essential references). In this publication we review groups the research on the use of essential oils and oils are the hydrocarbon terpenes and phenyl- several individual compounds found in propanes. Terpenes, the main components, with about 90 % of the are subdivided essential oils as control measures for V. jacob- total, soni. into monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20). Monoterpenes, 3. SCREENING the main essential oil terpenes, are volatile OF ESSENTIAL OILS and often appear with functional groups such as alcohols, phenols, aldehydes, In all, more than 150 essential oils and ketones, esters and oxide derivatives. The components of essential oils have been essential oil components thymol, eucalyptol, tested, but only the ones with a positive effect camphor and menthol have a FAO GRAS are mentioned in this section (for a complete status (generally recognized as safe) in con- list of all oils and chemical substances used centrations of up to 50 mg·kg-1. In pure see the references in tables I-III). form, however, these compounds, as well as many essential oils, can irritate eyes and mucous membranes and should be handled 3.1. Mite and bee toxicity with care. Further details about essential oils can be found in extensive monographs Hoppe [42] examined 55 essential oils on the subject [1, 37, 38, 79, 95]. for mite toxicity by evaporation and topical application tests. In the evaporation test, oil. The mortality of mites and bees was a group of ten mites, fixed dorsally on a evaluated after 24, 48 and 72 h. After 72 h, 24 essential oils a mite of small glass plate and placed in a 37-mL glass produced mortality more than 90 % After were exposed to 0.5 μL of pure essential oil (table I). topical application, where the maximal effect was applied on a filter paper. For the topical reached after 48 h, only three oils produced application test, mites were fixed in the same the same level of mite mortality. This sug- way, and 0.2 μL of a 5 % solution of essen- gests that passive evaporation is the most tial oil in acetone was to aqueous applied appropriate form of application for the the ventral side of the mites. to Toxicity essential oils and their components. Of the bees was evaluated 20 bees in a by placing 24 oils causing a high mite mortality, only small cage held in a closed 3.4-L glass nine resulted in bee mortality below 10 %. In receptacle containing 10 μL pure essential another bee toxicity test in which 1 mL of a 0.5-20 % essential oil solution in aqueous a glass petri dish. Mite mortality was 100 % acetone was sprayed on caged bees, only with oregano oil, 87.2 % with clove oil, elevated concentrations of wintergreen oil, 75.5 % with bay oil and 59.4 % with tea- which were well tolerated by bees, caused a tree oil. high mite mortality. From 55 essential oils Marchetti et al. examined the effects oil was chosen for field [68] only wintergreen of the of smoke trials. application produced by burning material from ten different plants. Kraus [53] investigated mite and bee Twenty smoke strokes from 4 g plant mate- mortality resulting from exposure to mar- rial were introduced into test hives. The joram, cinnamon, clove, citronella and entrance of the hives was closed for 60 min. lavender oils. Ten bees, carrying one mite Mentha piperita was the most efficacious, each, were kept under a beaker with a piece detaching 75 % more mites than obtained of wax foundation containing 0.1, 1 or 10 % with control smoke from combusted sack of an essential oil. After 3 days, both mite jute. Eischen and Wilson [28, 29] treated and bee mortality were determined. Clove small groups of approximately 250 infested oil at 1 % concentration in wax caused mite bees with cool smoke from burning plant mortality of more than 80 % and a bee mor- materials for 60 s and evaluated the mite tality equal to that in the untreated control, fall over 24 h. The smoke from Tanacetum while at 10 %, both mite and bee mortality vulgare, Juniperus virginiana, Citrus sp., were close to 100 %. Application of 10 % Larrea tridentata, Melaleuca quinquenervia, Oxalis rubra and Rhus marjoram oil resulted in a mite mortality of Azadirachta indica, 100 % and bee mortality of 20 %, the lat- typhina knocked down 70-90 % of the ter not being significantly different from the mites. control. Imdorf et al. [49] devised an assay in Bunsen [12] tested bee tolerance to laven- which the dose-response relationships der, melissa, wintergreen, fir needle, Pinus between an airborne acaricide and corre- mugo ssp. mugo and neem oils and citral. sponding mite and bee mortalities were In cages containing 20 bees, 300 μL of 0.1, examined for several volatile substances. 1 or 10 % oil in acetone were evaporated For each test, two cages (Liebefeld type), from a filter paper. Bee behavior was each containing 100 bees and 20-40 mites, observed for 7 h. All concentrations of cit- were placed in a desiccator and exposed to ral, but only the highest concentrations of air containing the vapor of specific volatile lavender and melissa oils, disturbed bee substances at specific concentrations. Dif- behavior. The other oils showed no effects. ferent concentrations were produced by mix- ing fresh air with air containing the active Colin et al. [19] sprayed water emulsions ingredient. The test was performed in an 1.25, 2.5, 5, 10, 20 or 40 containing g·L-1 incubator at a of 32 °C and a of and temperature thyme, sage, Chenopodium spp. relative of 50-60 %. The concen- Anona oils onto filter and humidity spp. paper placed tration of the active in the desic- them in a dish ten ingredient plastic petri containing cator was measured at and 72 and mites. After 24 emulsions 24, 48 h, h, containing after 72 h the dead bees and the fallen mites 20 g·L-1 of thyme, sage and chenopodium were counted. Concentrations of 5-15 μg oils which caused a mortality of more than of thymol, 50-150 μg camphor and 20-60 μg 90 % compared to less than 5 % for the menthol per liter of air killed 100 % water control. nearly of the mites without a noticeable loss of Sammataro et al. [83] exposed four to bees. Eucalyptol at 240 μg·L-1 caused 100 % five mites to the vapors of 40 μL of a 50 % mite mortality, but also killed 25 % of the solution of essential oil dissolved in olive bees. Imdorf et al. (unpublished data) tested oil and applied to filter paper discs placed in other monoterpenes using the same assay and found that concentrations of 400-1 000 μg after 72 h. Tables II and III summarize the p-cymene, 120-260 μg α-thuyon and results of those tests in which essential oils 30-100 μg isopinocamphon per litre of air and different natural substances (most of caused mite mortality of nearly 100 %, but them being components of essential oils) were well tolerated by bees. Isopinocam- exerted an effect in any of the tests. A direct phon is the main component of ysop oil comparison of the repellent and the attractant (isopinocamphon type). Mortality resulting effects in the different studies is not possible from exposure to α-terpinene was high for because the various tests were quite differ- both mites and bees. Limonene and α-pinene, ent. The effects are, however, mostly cor- even at high concentrations, produced little roborative, e.g. lavender oil was repellent mortality of either mites or bees. in all three tests where it was examined. Not all oils and organic substances showed effects in the different tests. 3.2. Repellent, attractant and effects on reproduction Kraus [53, 54] developed other tests to V. and brood jacobsoni tolerability evaluate the effects of essential oils on olfactory orientation by V. jacobsoni. Citronella, Different screening tests were used for lavender and marjoram oils exhibited repel- studying repellent and attractant effects. lent effects, while cinnamon and clove oils and a wax arena Hoppe Schley [44] placed were attractants. The capacity of V. jacob- divided into four parts in a petri dish. One soni to differentiate between drones and microliter of oil was at the of applied edge workers was not disturbed. The orientation two of the four opposite fields. Ten mites of V. jacobsoni towards adult bees and its were released in the middle of the arena and capacity to differentiate between nurse bees their locations were recorded 2 min. every and pollen foragers was impeded. When for 30 min. Kraus devised an con- [53] assay brood comb wax of a mating hive contained sisting of two small wax tubes (length = 0.1 % of the repellent marjoram oil, the 3 diameter = 1 = 5 The cm, cm, weight g). brood infestation of V. jacobsoni was sig- test substances were mixed in the wax of nificantly diminished. This phenomenon one of the tubes at concentrations of 0.1, was explained by a disturbance of V. jacob- 0.5 or 1 %, the other tube being an untreated soni brood recognition. control. Five mites were placed in the two connecting tubes, their position was Bunsen [12] conducted field trials with observed through the thin wax tube wall oils from fir needles, Pinus mugo spp. mugo, after 30 and 90 min. Bunsen [12] developed lavender, melissa, wintergreen and with cit- a test where 10 μL of a 1 % emulsion of ral, all of which, with the exception of win- active ingredient in water were dropped on tergreen, were repellent in the screening test brood in cells just before operculation. A (table II). One comb from a bee colony was control treatment was conducted with 10 μL sprayed with 20 mL acetone-oil solution water containing 1 % emulsifier. The brood (9:1 vol/vol) and the same variables were was frozen 18 days after egg laying, and the examined as in the screening test above. following variables were measured: brood Only the attractant effect of wintergreen oil mortality, repellent and attractant effects was confirmed. This effect, however, and effect on mite reproduction. Colin et al. decreased with increasing mite infestation [19] devised a choice test using a petri dish rate. Unexpectedly, lavender oil, which was arena in which mites had a choice between repellent in the laboratory test, exhibited an a bee larva sprayed with an emulsion of attractant effect in the field trial and caused 20 g·L-1 of active ingredient or water as a a higher mite infestation rate of brood. Also, control. The distribution of mites between both Pinus mugo spp. mugo oil (without an treated and control larvae was determined effect in the screening test) and fir needle oil (repellent in screening test), significantly with shoots). One colony group was fed decreased mite reproduction. However, 30-60 mL of a decoction-sugar solution, when all combs of a small mating hive were the other was treated with Folbex. Average sprayed with the same oils, these effects mite drop per colony was 1 682 and 1 489, could not be reproduced. The effectiveness respectively, in each group. The percentage of trapping combs treated with the attrac- mortality was not determined. tive oils of lavender and for wintergreen After extended laboratory of mite was no screening reducing populations greater essential oils, evaluated the than that of the untreated controls. Hoppe [42] effect of the passive evaporation of 5, 10 Summarizing the results of all different and 15 mL of clove and wintergreen oil from tests, it can be concluded that the effects soft cardboard in normal colonies. Evapo- observed in the different tests are not con- ration of 5 mL of either oil produced no sistent and that it is difficult to predict the effect. Evaporation of 10 mL of clove oil varroacidal activity of essential oils in field produced mite and bee mortalities of 47 and conditions from their performance in labo- 27 %, respectively, while evaporation of The behavior were ratory assays. tests aimed 10 mL of wintergreen oil resulted in mor- at developing a method to disturb olfactory talities of 72 and 2 %, respectively. Evapo- orientation of V. jacobsoni. When the tests ration of 15 mL of clove oil produced mite were carried out with small colonies, how- and bee mortalities of 92 and 50 %, respec- most oils and ever, chemical substances tively, compared to 95 and 7 %, respec- were not efficient in reducing mite popula- tively, with 15 mL of wintergreen oil. Both tion. Only marjoram, sage, thyme and win- higher dosages led to behavioral distur- tergreen oils turned out to have an acceptable bances of bees. Later, Hoppe and Ritter level of activity against V. jacobsoni in bee found that two combined treatments of colonies (see section 4). brood-free colonies consisting of 5 mL of wintergreen oil (methyl-salycilate) and a 15-min thermal treatment of 54 °C [11, 43] 4. APPLICATION IN THE HIVE resulted in a mite mortality of 93 % and were well the bees. A similar 4.1. Essential oils supported by combination of treatments, but with a 30-min thermal treatment in colonies with brood, The first trials for the control of V. jacob- caused 31 % mortality of adult mites after soni with essential oils in free flying colonies 5 days and blocked the reproduction in 69 % were conducted in Russia. Sidorov et al. of the mites, to 23 % infertile tested the effects of compared [86] mint, pine-needle, mites before treatment [13]. aniseed, fennel, wormseed, dragons head, citralinic and Chenopodium oils. All tested Neem oil from seed-kernel showed a oils had an unquantified activity against repellent effect and a high brood mortality under conditions V. jacobsoni. Wormseed oil had a relatively laboratory [12]. However, low efficacy, while Chenopodium oil was passive evaporation of 10 g of neem oil very toxic for bees. Treatment of bee every 3 weeks from a petri dish from below colonies caused excitation of bees. the brood from the middle of July until the beginning of winter, had no effect on bee Shutov [85] made a decoction out of a and mite populations [12]. mixture of leafless Anabasis sp. (shoots), Calendula sp. (herbs), Tanacetum vulgare Colin [18] compared an aerosol applica- (flowers), Leonurus cardiaca (herbs), Matri- tion of water emulsions of 1 % thyme oil caria recutita (herbs), Ephedra sp. (shoots), (main ingredients p-cymene and thymol) Quercus sp. (roots), Artemisia apsithicum and 0.5 % sage oil (main ingredients: cam- (shoots, inflorescences) and Pinus sp. (buds phor, α-thujone and eucalyptol) with the application of a 0.25 % Amitraz solution in of 4.5 L of 15 % formic acid). At the same colonies having a small amount of brood. time seven treatments of marjoram oil, each The aerosol treatment applied with warm consisting of 3 mL oil applied on two air through the flight hole was carried out wooden strips on top of the brood chamber, for 1 min and was repeated four times at were made (total of 21 mL of marjoram oil). intervals of 3-4 days. The mixture of thyme In Vietnam, the same application with a and sage oils resulted in 95 % mite mortal- treatment period of only 24 days and appli- ity compared to 99 % mortality obtained cation intervals of 3 days, showed 98 % effi- with Amitraz. This mixture was commer- ciency. No brood and bee losses were cialized under the name of BIOLOGIC V®. observed. However, this method is very Liebig [57] tested this product in brood-free material and time-consuming. colonies in November. Four, 1-min treat- Other treatments with essential oils have ments at intervals of 3-4 with 100 mL days been reported in the apicultural press and of BIOLOGIC V® 1.5 L deionized water per trade-journals, e.g. the use of Japanese were carried as described out, essentially medicinal plant oil [71] and the use of win- Colin Mite was about by [18]. mortality tergreen oil, alone or in blends with linseed, 10 %. The different efficiencies in these two pennyroyal, spearmint or tea-tree oil, and trials was explained as a possible conse- blends of patchouli with spearmint oil [2, different of quence of compositions the 78]. However, most of these trials were not essential oil used and serves to illustrate the carried out under standardized test condi- problems arising from not standardizing the tions and the efficacy of these treatments essential oils, as previously mentioned. The remains uncertain. thymol content of thyme oil of different ori- gins and chemotypes varies between 5 and 40 % [20]. Under the subtropical conditions 4.2. Components of essential oils of the coastal plain of Israel, Gal et al. [34] treated colonies in spring with 20 % solution 4.2.1. Thymol of oregano oil. Colonies with brood occu- pying one super were treated for 25 days Thymol is the only essential oil compo- in consis- with a piece of cardboard (35 x 50 x 0.2 cm) nent widely used apiculture that soaked with a solution of 50 mL of oregano tently exhibits high varroacidal activity and was first oil (20 and 33 % in a 1:1 vol/vol ethanol- that is well tolerated by bees. It used A. woodi paraffin mixture). The cardboard was placed against [52]. Mikijuk [73] tested the varroacidal of in on the top of the brood chamber. A 2nd activity thymol bee colonies. He used 0.25 in piece of cardboard was placed on the bottom g per space board in colonies with two hive bodies. The between combs and obtained mite mortality of 55 %. found that while average mortality was 82 %, while with Mikijuk [72] thymol increases with 33 % solutions, mortality was 91 %. Treat- evaporation greatly ments at higher temperatures produced increasing temperature or evaporation area, it was not influenced air It was harmful effects such as robbing and by humidity. recommended that weak colonies should absconding. Therefore, this method was rec- not be treated when are ommended as a spring treatment during the temperatures greater citrus honey flow. Le Tu Long [55] obtained than 27-30 °C. 99 % mite mortality with a combined treat- Impressed by the widespread use of ment of marjoram oil and 15 % formic acid thymol in combination with drone brood for 28 days. Seven treatments of 15 % removal in Yugoslavia, Marchetti et al. [69] formic acid, the first one being 1.5 L and tested the application of 15 g powdered thy- the subsequent ones 0.5 L each, were applied mol suspended in a small gauze bag between on the bottom board at 4-day intervals (total two outer brood combs (table IV). Other studies with powdered thymol employing [50, 59, 84]. When evaporation rate, and different quantities and application inter- hence dosage, are increased, the efficacy is vals during different seasons of the year also increased [15, 84]. Efficacy also have been conducted by workers in Italy increases with increased treatment duration and Spain ([17, 31, 33, 39, 40, 62], see [50]. Treatments applied at the bottom of table IV). The average mite mortality varied hives produced insufficient control [50, 59]. from 66 to 98 %. In of the differences spite All components do not contribute to the in the methods, mite mor- application high varroacidal activity of Api Life VAR®. The tality combined with low colony-to-colony presence of camphor had no influence on was observed. [60] and variability Liebig the treatment 84, Bollhalder melted and efficiency [50, 91]. ]. Also, [ 10] thymol poured the concentration of menthol and eucalyptol the solution on a viscose sponge, which was in the hive [50] was much lower than nec- subsequently placed on the brood combs. essary for good mite toxicity [49], while the The was to that of efficiency comparable thymol hive concentration measured has the (table IV). Marchetti powder application caused 100 % mite toxicity in laboratory et al. [69] and Lodesani et al. [62], how- trials section we can assume have low with (see 3.1). Thus, ever, reported mortality thy- that is the active of this mol. thymol ingredient formulation. This conclusion is supported Knobelspies [51] developed a continu- by other trials [10, 60] where treatments ous treatment method with thymol. A small with pure thymol, applied under similar con- chamber is placed between the brood combs, ditions and with comparable amounts to the and thymol evaporates through evaporation ones used with Api Life VAR®, resulted in slots. Twelve grams of thymol are supplied a comparable efficacy. in May and again in August. Although there Low mite has been are no published results on trials carried out mortality reported under controlled conditions, anecdotal with this product in some studies. This has been as a result of either a treat- reports on the long-term use of this method explained ment of insufficient duration or of claim that V. jacobsoni populations are period maintained below economic injury levels, low temperatures that do not allow for adequate (table V). Also, consid- provided that there is no mite re-invasion. evaporation erable colony-to-colony variability in effi- even within the same has been 4.2.2. Api Life VAR® cacy, apiary, observed. Therefore, due to the limited reli- of the it has been Life VAR® LAIF, ability product, suggested Api (Chemicals Italy) that it be used in combination with other is composed of a porous ceramic carrier measures such as drone brood for- (florist block material 5 × 9 × 1 cm) impreg- removal, mation of nucleus colonies, and the appli- nated with a mixture of thymol (76 %), euca- cation of oxalic acid or other varroacides menthol and cam- lyptol (16.4 %), (3.8 %) brood-free phor (3.8 %). The vermiculite tablet is laid during periods [46-48, 65, 77]. The same conclusion applies to the other on the upper part of the brood combs. After thymol treatments. Further research, how- 2-4 weeks of application, a second tablet is ever, may help define the conditions under placed in the hive for the same period. In which treatments can be relied 14 of 22 15, 22, 35, 46, thymol upon applications ([ 14, for control of 50, 59, 65, 76, 77, 80, 84, 88, 91], Mutinelli adequate V. jacobsoni. et al., unpublished data) mite mortality was During treatments with Api Life VAR®, greater than 90 % (table V). At normal bees remove feed and brood in the vicinity depth, one-story bee hives, a higher effi- of the applied product. Also, some colonies cacy was achieved than in larger (oversized) have difficulties taking supplementary feed one-story hives and in multiple-story hives during the treatment period [46, 59, 76, 80,

84]. Therefore, winter feed, when neces- icantly different from the control. The effi- sary, is best given before the treatment. cacy of camphor was 71.9 %. The results Moosbeckhofer [76] observed negative of the laboratory trials of Imdorf et al. [49] effects of Api Life VAR® treatments on showed that menthol and camphor caused colony development, as well as stimulation a high mite mortality at concentrations of robbing and increased aggressiveness. which were not toxic for bees. Thus, fur- The first two effects mentioned were not ther trials to develop the optimal applica- reported in other studies. tion mode are necessary to clarify whether this substance can successfully be used for 4.2.3. Other thymol blends mite control. Two applications of 17 g of linalool applied 14 days apart to brood-free Calderone et al. [ 16] evaluated the appli- colonies produced a mite mortality of only cation of different thymol blends (1:1 wt/wt) 27.4 % [15]. The reason for this low effi- be due to the weak varroacide on colonies in three different apiaries. cacy may Blends included thymol and linalool, thy- effect of this substance or it may require mol and eucalyptol, and thymol and cit- higher temperatures for a better evaporation ronellal. Two application rates, 25 and 12.5 g/ and effectiveness. application, were tested. The first application was replaced by a second after 14 days. 5. RESIDUES OF ESSENTIAL OILS Total treatment period was 28 days in the late-summer and autumn of 1994. They achieved an average mite mortality in the Essential oils, and components thereof, thymol-eucalyptol treatment groups of 56 have been used with varying degrees of suc- and 49 % for the higher and lower rates, cess for mite control. However, there are respectively. Corresponding values were 43 few studies dealing with residues in honey and 38 % for thymol and citronellal, and 40 and other hive products, and those that have and 30 % for thymol and linalool. Rate been carried out were performed after the effects were not statistically significant for use of individual essential oil components, any of the three blends. The overall low rather than after treatment with essential level of mortality was attributed to the large oils. Since essential oils are, themselves, amounts of brood present during treatment, complex blends of compounds, and since as a much higher efficacy of 96.7 % was most honeys naturally contain many essen- achieved with the application of a similar tial oil constituents as aroma components thymol blend on nearly broodless colonies [67], analysis of residues after treatment [15]. with essential oils can be complicated and inconclusive. This can make compliance 4.2.4. Menthol, camphor and linalool with EU and US governmental regulations difficult. Sidorov et al. [86] reported an unquanti- Residues are a significant concern, not fied acaricidal activity of menthol in bee only because of possible health considera- colonies, in addition to that of other essen- tions, but because of their possible effects on tial oils. Menthol was also tested by Mik- honey quality. According to EU regulation itjuk et al. [73]. Its efficacy was only 13 %, no. 2377/90, thymol is in group II of the that of thymol was 55 % (0.25 g per space in non-toxic veterinary drugs, which do not between combs used). Higes et al. [41] need a MRL (maximal residue limit). Essen- treated two groups of four broodless colonies tial oils are, however, odor-intensive sub- over 4 weeks with either 30 g of menthol stances and very small amounts in honey or 60 g of camphor. Menthol had an average can alter its taste. When thymol is used for efficiency of 20.5 %, which was not signif- control of V. jacobsoni when there is no nectar flow in progress, residues found in these chemicals. Essential oils, and espe- honey vary from 0.02 to 0.48 mg·kg-1 [6, cially components of essential oils, may 77, 64, 90]. However, if thymol is used dur- serve as alternatives or as adjuncts to tradi- ing nectar flow, there is considerable danger tional treatment measures. In extensive that residues may reach levels above the screening tests, many oils show significant taste threshold. Bogdanov et al. [8] found acaricidal activity. Some oils are repellent to that 1.1-1.6 mg of thymol·kg-1, 5-10 mg·kg-1 V. jacobsoni, others are attractive, and some of camphor and 20-30 mg·kg-1 of menthol cause mite mortality. However, very few of can significantly alter honey taste. For this them have proven effective when applied reason, in Switzerland a MRL value of in hives in field trials. Considerable variation 0.8 mg thymol·kg-1 was fixed. The treat- in local environmental and colony condi- ment method of Knobelspiess [51] is used in tions affect efficacy and make it difficult to different European countries. This method predict the outcome of many treatments. delivers thymol in the hive continuously Difficulty in obtaining standardized essen- throughout the entire year. Honeys harvested tial oils also affects treatment predictabil- in bee hives treated with this method often ity. Only a combination of wintergreen oil have thymol residues higher than the Swiss and thermal treatment, an aerosol treatment MRL value of 0.8 mg·kg-1 [7]. of a thyme-sage oil mixture, and the passive evaporation of thymol, oregano oil and Bogdanov et al. [6], evaluated the effects marjoram oil in combination with diluted of a long-term use of Api Life-VAR® (main formic acid have been successfully used for ingredient thymol) on honey and wax mite control (see sections 4.1 and 4.2). For residues. The residues in and thymol honey several reasons, however, none of these wax did not increase with num- increasing treatments have been widely adopted by ber of Api Life-VAR® treatments. Brood beekeepers, with the exception of thymol. comb residues reached a level relatively high Indeed, thymol and thymol blends are (average of 517 mg·kg-1). However, if Api widely used to control V. jacobsoni in Life VAR® treatment is stopped, thymol Europe and in most cases their varroacidal rapidly evaporates from wax and after 1 year efficacy is greater than 90 %. In due time, drops to near the limit of detection. In different thymol-containing products will bottled unopened, honey, thymol, camphor be available on the market. In laboratory and menthol remain stable for 2 years [6]. tests, a high mite toxicity, combined with bee were demonstrated for Menthol, another substance with a var- good tolerance, a number of other of essential roacidal activity [49], has been used for con- components oils trol of A. woodi in North America. After a (see section 3.1). 3-week treatment, a maximum of 18 mg·kg-1) Identifying compounds with acceptable in honey and 2 790 mg·kg-1) in wax were acaricidal activity but with low toxicity to detected [56]. This level is below the sensory honey bees is essential for providing can- threshold of menthol in honey. didate compounds for field trials. Future research can assist in this effort by focus- ing on the characterization of the dose- 6. DISCUSSION response relationships between essential oils and 1) mite/bee toxicity, and 2) effects on mite behaviour. This can serve New alternatives for the control of procedure as a because V. are necessary because of the powerful screening technique jacobsoni it field research into the rapid and widespread development of guides subsequent pyrethroid and organo-phosphate-resistant most productive avenues. mite populations and because of the poten- The development of effective delivery tial for contamination of hive products by systems for essential oils remains one of the greatest roadblocks to their implementation out of drone brood, trapping combs, forma- as mainstream control measures. Highly tion of nucleus colonies or the use of organic volatile substances such as camphor are dif- acids [47, 48, 58, 82, 81, 94], into an inte- ficult to use, but formulations retarding the grated pest management strategy for the evaporation rate, e.g. special gels, might control of V. jacobsoni. Adapting these overcome this difficulty. Products with mix- strategies to local climatic conditions, to tures of different components with differ- diverse apicultural management practices ent modes of action, might also provide and to beekeeping operations of varying effective solutions. For example, substances sizes pose additional and significant chal- that disrupt the mite’s host location process lenges. Finally, resistance to essential oils may be effective in conjunction with sub- may eventually develop, as it has with syn- stances that kill mites. thetic pesticides. Consideration must be given to the development of resistance man- Residues another to the pose challenge agement plans to maximize the useful life use of essential oils. Most essential oils are span of effective acaricides and delivery mixtures of more than 50 components. systems once they are developed. Depending on the individual partition coef- ficients of the constituents, residues in honey and wax are to be expected. Residues in ACKNOWLEDGEMENT honey can lead to adverse effects on taste, while residues in wax can render it unsuitable This review was supported in part by a grant for some applications. Quantitative residue from the USDA-CSREES Northeast-IPM Pro- analyses are required for product registra- gram (#9704078) to NWC and by a grant from tion. The complex nature of many essential the NYS Department of Agriculture and Mar- kets to NWC. oils, combined with the fact that many essen- (#C970001) tial oil components are naturally occurring in honey, makes such residue analysis difficult. Thus, the successful development of Résumé - Utilisation des huiles essentielles dans la lutte contre products employing essential oils can be Varroa jacob- extremely difficult unless the particular soni, parasite des colonies d’abeilles. Le de la résistance des essential oil has been granted an exemption développement popu- from tolerance. The use of individual com- lations de Varroa jacobsoni et le spectre de ponents of essential oils makes residue anal- la contamination des produits du rucher ont ysis much easier and limits the potential for fortement stimulé la mise au point de nou- producing off-flavor honey. Long-term stud- velles stratégies de traitement qui diminuent ies demonstrated that when used properly, la possibilité d’un développement rapide de residues of thymol in honey remain at low la résistance et l’accumulation de résidus. and safe levels. Cet article passe en revue l’utilisation des huiles essentielles (h.e.) et de leurs compo- Based on the available studies, relying sants dans la lutte contre V. jacobsoni. solely on one treatment per bee season with « Huiles essentielles » est un terme géné- essential oils or essential oil components rique qui désigne les composants liquides can not be recommended as an effective and et hautement volatiles des plantes, marqués reliable method to maintain mite popula- par une odeur forte et caractéristique. Les tions below the economic injury level. The terpènes (principalement les monoterpènes) challenge for future research is to optimize représentent la majeure partie (environ 90 %) the use of essential oils and essential oil de ces composants. L’eucalyptol, le camphre, components and to incorporate the result- le menthol et le thymol sont des monoter- ing products along with other measures for pènes typiques. Chaque espèce de plante limiting mite populations such as cutting tend à avoir une composition en h.e. unique. Pourtant, certaines espèces ont des variétés comme sans danger), de la FAO aux concen- dénommées chémotypes, dont la composi- trations allant jusqu’à 50 mg·kg-1. Les rési- tion en h.e. varie. On a utilisé les méthodes dus de ces substances dans le miel ne posent de criblage au laboratoire pour tester sur V. donc pas de problème toxicologique. Après jacobsoni et sur les abeilles la toxicité, la un traitement au thymol les résidus dans la répulsivité, l’attractivité, ainsi que les effets cire sont 1 000 fois plus élevés que dans le sur la reproduction, des h.e. et de leurs com- miel, mais ils diminuent rapidement par éva- posants. Au total, c’est plus de 150 h.e. et poration dès que le traitement est arrêté. composants d’h.e. qui ont été testés (tableaux D’après les études disponibles, un seul trai- I-III), mais peu d’entre eux se sont montrés tement à base d’h.e. ou d’un composant efficaces lors de leur utilisation sur ruches d’h.e. est généralement insuffisant pour en conditions de terrain. Une variation maintenir la population d’acariens en des- énorme des conditions du milieu local et sous du seuil de dégât économique pendant des colonies, ainsi que la difficulté d’obte- toute la saison. Des efforts sont donc néces- nir des h.e. standardisées, rendent difficile la saires pour intégrer ces produits, ainsi que prévision du résultat des nombreux traite- d’autres mesures pour limiter les popula- ments. Les résidus dans le miel peuvent tions d’acariens tels que l’élimination du conduire à des effets négatifs sur le goût. couvain de mâles, les rayons pièges, la for- L’analyse quantitative des résidus est exi- mation de nuclei et l’utilisation d’acides gée pour l’enregistrement du produit. La organiques, dans une stratégie de lutte inté- nature complexe de beaucoup d’h.e. et le grée contre V. jacobsoni. © Inra/DIB/AGIB/ fait que de nombreux composants des h.e. Elsevier, Paris sont présents à l’état naturel dans le miel / / lutte font que l’analyse des résidus est difficile. Apis mellifera Varroa jacobsoni / huile essentielle / résidu Néanmoins ces difficultés disparaissent chimique lorsqu’on utilise individuellement les composants des h.e. La plupart sont volatils et disponibles sur le marché à prix raison- Zusammenfassung - Ätherische Öle zur nables. Parmi tous les composants testés des Bekämpfung von Varroa jacobsoni in h.e., c’est le thymol qui a eu le meilleur Honigbienenvölkern. Die Entwicklung von résultat en apiculture pratique. Dans les Varroazidresistenzen und das Ausmaß ver- essais en champ le thymol pur (tableau IV) schiedenartigster Verunreinigungen von Bie- et les mélanges à base de thymol (tableau V) nenprodukten stellt eine dringende Heraus- ont montré une activité varroacide élevée. Le forderung zur Entwicklung neuer Behand- thymol est bien toléré par les abeilles. Si les lungsstrategien dar, durch die diese Nach- traitements sont effectués en dehors de la teile eingegrenzt werden können. Der vor- période de miellée, les résidus de thymol liegende Artikel gibt einen Überblick über dans le miel n’augmentent pas lorsqu’on die Nutzung von ätherischen Ölen oder ihrer multiplie les traitements et restent sous le Komponenten zur Bekämpfung von Varroa seuil de détection gustative, qui se situe entre jacobsoni. Als ’ätherische Öle’ werden hier- 1,1 et 1,6 mg·kg-1. Selon les études on a bei flüssige, hochflüchtige Pflanzenbe- trouvé des résidus compris entre 0,02 et standteile zusammengefasst, die durch inten- 0,48 mg·kg-1. Mais les traitements au thymol siven und charakteristischen Duft gekennzeich- pendant la miellée peuvent conduire à des net sind. Den Hauptanteil stellen mit etwa résidus plus élevés qui modifient le goût du 90 % Terpene, zumeist Monoterpene. Typi- miel. Le thymol, de même que d’autres sche Monoterpene sind zum Beispiel Euka- monoterpènes tels que le menthol et le lyptol, Kampfer, Menthol und Thymol. Die camphre, ont le statut GRAS (generally Mischung ätherischer Öle ist zumeist für recognized as safe, généralement reconnu die jeweilige Pflanzenart einheitlich. Aller- dings bilden einige Pflanzenarten Varietäten gefunden. Allerdings können Thymolan- aus, sogenannte Chemotypen, die sich in wendungen während der Trachtzeiten zu der Zusammensetzung ihrer ätherischen Öle höheren Rückständen und verändertem unterscheiden. Geschmack des Honigs führen. Thymol Toxische Eigenschaften oder die Wirkun- sowie andere milbengiftige Monoterpene gen als Attraktans, Repellent oder auf die wie Menthol und Kampfer werden bis zu Reproduktion wurde in Labortests unter- 50 mg·kg-1 generell als sicher angesehen sucht. Insgesamt wurden hierbei bisher über (Status FAO GRAS ). Rückstände dieser 150 verschiedene ätherische Öle oder ihre Substanzen im Honig sind daher toxikolo- Komponenten getestet (Tabelle I-III). Aller- gisch ohne Belang. Thymolrückstände im dings erwiesen sich nur wenige als wirk- Wachs sind nach einer Behandlung um sam, sobald sie in Feldversuchen in Völ- einen Faktor von 1 000 höher als im Honig, kern angewandt wurden. Die erheblichen nach der Anwendung vermindern sie sich Unterschiede in den örtlichen Umgebungs- allerdings rasch durch Verdunstung. bedingungen und zwischen den Völkern Nach den bestehenden Untersuchungen ist beeinflussen die Wirksamkeit und erschwe- eine einzige Behandlung mit ätherischen ren eine zuverlässige Aussage über den zu Ölen generell nicht ausreichend, um die Mil- erwartenden Behandlungserfolg. Hinzu benpopulation über eine ganze Saison unter- kommt die Schwierigkeit, standardisierte halb der ökonomischen Schadensschwelle ätherische Öle zu erhalten. Die Rückstände für die Bienenvölker zu halten. Zur Begren- im Honig können den Geschmack negativ zung der Milbenpopulation sind daher im beeinflussen. Zur Registrierung eines Rahmen einer integrierten Bekämpfungs- Behandlungsmittels sind quantitative Rück- strategie zusätzliche Maßnahmen erforder- standsanalysen erforderlich. Diese wird lich, wie das Ausschneiden von Drohnen- durch die komplexe Zusammensetzung vie- brut, Fangwaben, Jungvolkbildung oder die ler ätherischer Öle und durch die Tatsache Anwendung organischer Säuren. © Inra/ erschwert, daß viele der Komponenten auch DIB/AGIB/Elsevier, Paris natürlicherweise im Honig vorkommen. Die Nutzung einzelner Komponenten der äthe- Apis mellifera / Varroa jacobsoni / rischen Öle ist allerdings nicht von diesen ätherische Öle / Wirksamkeitstests / Schwierigkeiten begleitet. Die meisten Rückstände Komponenten sind flüchtig und zu annehm- baren Preisen auf dem Markt erhältlich. In der praktischen Imkerei hat sich von allen REFERENCES untersuchten Komponenten ätherischer Öle Adams Identification of Essential Oils am besten bewährt. In Freiland- [1] R., by Thymol Ion Trap Mass Spectroscopy, Academic Press, versuchen zeigten Thymol (Tabelle IV) und 1989. Thymolmischungen (Tabelle V) eine hohe [2] Amrine J.W., Stansy T.A., Skidmore R., New varroazide Wirksamkeit. 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