T urk. J. Vet. Anim. Sci. 2012; 36(5): 554-559 Research Article © TÜBİTAKdoi:10.3906/vet-1104-12 Varroacidal effi cacies of essential oils extracted from Lavandula offi cinalis, Foeniculum vulgare, and Laurus nobilis in naturally infested honeybee (Apis mellifera L.) colonies Figen KÜTÜKOĞLU1, Ahmet Onur GİRİŞGİN2,3,*, Levent AYDIN2,3 1Directorship of Food, Agriculture, and Animal Husbandry, Bursa - TURKEY 2Department of Parasitology, Faculty of Veterinary Medicine, Uludağ University, Bursa - TURKEY 3Beekeeping Development-Application and Research Center, Uludağ University, Bursa - TURKEY Received: 19.04.2011 ● Accepted: 27.12.2011 Abstract: Th is study was performed to determine the effi cacies of Lavandula offi cinalis, Foeniculum vulgare, and Laurus nobilis essential oils on the control of Varroa destructor (Acari: Varroidae), the most common parasite of honeybees (Apis mellifera L.). Experimental colonies from the province of Bursa, Turkey, were divided into 5 homogeneous groups of 8 hives each. Th e 3 essential oils plus thymol (Th ymovar®, an essential oil-based drug with known effi cacy that was used for comparison) were applied to 1 group each and 1 group was left untreated as a control. Each group was treated during 3 seasons, 2 consecutive autumns and 1 spring. Effi cacies of the essential oils were determined by the Henderson- Tilton formula and the signifi cance between the oils was determined by Tukey’s multiple comparison test. In the fi rst autumn, the highest effi cacy was detected in Th ymovar (79.4%) followed by L. nobilis (76.7%), L. offi cinalis (76.4%), and F. vulgare (74.5%) essential oils. In spring, the effi cacy rates were 83.8% in L. offi cinalis, 81.8% in Th ymovar, 78.8% in F. vulgare, and 70.8% in L. nobilis. In the second autumn, the effi cacy rates were 78.4% in Th ymovar, 76.6% in L. offi cinalis, 71.9% in F. vulgare, and 65.2% in L. nobilis respectively. No abnormal deaths were seen in adult bees during the treatment period. Key words: Effi cacy, essential oils, honeybee, Varroa destructor, Turkey Introduction initially named Varroa jacobsoni, but it was later Th e ectoparasitic mite Varroa destructor is a pest of identifi ed genetically (4) and morphologically (5) as the honeybee Apis mellifera. Colonies infested with V. destructor in Turkey. V. destructor have signifi cantly reduced worker bee Several synthetic acaricides have been applied populations and bees eventually die if left untreated to hives in order to fi ght varroosis but, due to the (1,2). widespread misuse of chemical treatments, several V. destructor came to Turkey from the Th race cases of resistance and contamination of hive region in 1977 and was spread across the country by products have been reported (6,7). Use of synthetic package bees in a short time, causing 600,000 colony acaricides causes accumulation in apiary products deaths (3). As in other parts of world, the agent was such as beeswax, propolis, royal jelly, and honey (8,9). * E-mail: onurgirisgin@gmail.com 554 F. KÜTÜKOĞLU, A. O. GİRİŞGİN, L. AYDIN Natural products such as essential oils off er a sunfl ower oil to obtain a 25% homogeneous mixture highly desirable alternative to synthetic products. of each. Impregnated onto the 5 × 5 cm felts (a Products of botanical origin have shown a wide range texture made of compressed matted animal fi bers) of biological activities including toxicity, repellence, was 10 cm3 of each essential oil (7.5 cm3 sunfl ower and antifeedant eff ects in parasites and growth oil + 2.5 cm3 essential oil). Each felt was covered with regulatory properties (10). plastic nets to prevent bees from coming into direct Bursa Province of Turkey is in the Marmara contact with and eating the felt, and aft erwards they Region, which has a quite humid (73% annual were covered with zipper bags and put into a freezer average) climate. Average minimum and maximum to prevent evaporation before application. temperature ranges in September, October, March, Experimental colonies and April are 13.5-27.0, 9.9-21.6, 3.5-13.6, and 7.2- Th ere were 40 Apis mellifera colonies used, which 18.7 °C, respectively (11). were highly infested with Varroa destructor. Th e Th e objective of this work was to determine the Langstroth-type hives, with 6-7 combs and large-size effi cacies of the essential oils of Lavandula offi cinalis pollen drawers, were placed in İkizce village in the (lavender), Foeniculum vulgare (fennel), and Laurus Nilüfer district of Bursa, Turkey. All colonies had nobilis (bay laurel) against Varroa destructor in the been left untreated for Varroa during the preceding province of Bursa, Turkey. Th ymol (Th ymovar®), a 12 months. Th e colonies were divided into 5 drug with known effi cacy, was used as a control to homogeneous groups with 8 hives in each group. compare these essential oils. Treatment and assessment procedures Th e colonies were treated in 3 seasons: September Materials and methods to October 2006 (fi rst season), March to April 2007 Characterization and preparation of essential oils (second season) and September to October 2007 Oils were purchased from Mecit Efendi Ltd. Şti. (third season). Th e average temperatures at the (İzmir, Turkey), derived from the leaves of each application times were 21.1 °C in September 2006, plant. Th e characterization of the 3 essential oils was 17.4 °C in October 2006, 13.5 °C in March 2007, 15.9 performed at the Department of Pharmacognosy, °C in April 2007, 22.0 °C in September 2007, and 18.2 Faculty of Pharmacy, Anadolu University, Eskişehir, °C in October 2007. Turkey, in August 2006. Doses consisting of 25% of Lavandula offi cinalis, Th e compositions of the oils were analyzed by Foeniculum vulgare, and Laurus nobilis essential oils ® gas chromatography-mass spectrometry (GC/MS), and thymol (Th ymovar ) were applied to each group; using an Agilent 5975 chromatograph equipped 1 group served as the untreated control. Th e control with an HP-Innovax fused silica capillary column group was divided into 2 subgroups; 1 subgroup had (60 m × 0.25 mm, fi lm thickness of 0.25 μm). Th e only sunfl ower oil in the felt and the other subgroup temperature was programmed to hold at 60 °C for 10 was left blank to understand whether sunfl ower oil min and then to rise to 220 °C at 4 °C/min, and the had an eff ect on the treatment. fi nal temperature was held for 10 min; injector and Approximately 200 adult worker bees from detector temperatures were 250 °C, the detector was a outer frames of each hive were collected into jars fl ame ionized detector (FID), and the carrier gas was containing cotton with diethyl ether to determine the helium at a fl ow of 0.8 mL/min. Th e identifi cation of rate of Varroa infectivity before and aft er treatment components was based on comparison of their mass (13). Aliquoted adult bees and mites were counted. spectra with those reported in literature (12) and by Additionally, the bottoms of the drawers were cleaned a computer search of their 70 eV mass spectra with before the trial and were covered with white paper to those stored in the library of the GC/MS data system, count dead mites that dropped into the drawer aft er as well as by retention indices. each drug application. Lavandula offi cinalis, Foeniculum vulgare, and Th e chosen colonies were treated with essential Laurus nobilis essential oils were diluted with oil-impregnated felts on days 1, 14, and 28 (once 555 Varroacidal effi cacies of essential oils extracted from Lavandula offi cinalis, Foeniculum vulgare, and Laurus nobilis in naturally infested honeybee (Apis mellifera L.) colonies every 2 weeks, totaling 3 applications). Only thymol Th e effi cacies of essential oils are presented in (Th ymovar®) was applied twice, as instructed, on days Table 2. Th e average effi cacies of all 4 products were 1 and 21. at similar levels, but L. nobilis essential oil had a lower In each season, the dead mites that had dropped effi cacy compared to the others. Th e commercial into the pollen drawers were counted on days 1, 3, 5, thymol extract on average showed the best effi cacy 7, 14, 21, 28, 35, and 42. Mites on bees were counted at the end of the 3 seasons (Table 2). Although an on day 42 aft er treatment. aggressive behavior was observed during the fi rst Statistical analysis minutes of applications, no abnormal bee mortality was observed during treatment. Th e effi cacies of essential oils were measured with the Henderson-Tilton formula. Signifi cance between the essential oils and signifi cance between the counts Table 2. Effi cacy of essential oils and thymol preparation on of dead mites per hive were determined via Tukey’s varroosis by Henderson-Tilton formula in 3 seasons. multiple comparison test at the level of 0.05 (14,15). Treatment Season Effi cacy % n in Co before treatment × n in T aft er treatment 1 76.4Lavandula offi cinalis 2 83.8 Corrected % = (1 – ) × 100 3 76.6 n in Co aft er treatment × n in T before treatment Average total 78.9 Where n = mite population, T = treated, Co = control. 1 74.5 Foeniculum vulgare 2 78.8 3 71.9 Results Th e main chemical components of the essential oils Average total 75.0 evaluated by GC/MS are presented in Table 1. For L. 1 76.7 offi cinalis the main compounds were α-pinene and Laurus nobilis 2 70.8 linalool; for F. vulgare they were (E)-anethole and 3 65.2 limonene; and for L. nobilis they were 1,8-cineole Average total 70.9 (eucalyptol) and α-terpinyl acetate (Table 1). 1 79.4 Th ymol (Th ymovar®) 2 81.8 Table 1. Composition of the 3 principal essential oils of 3 78.4 Lavandula offi cinalis, Foeniculum vulgare, and Laurus nobilis. Average total 79.8 Essential oil Compounds Composition%* α-pinene 49.4 Th e mean numbers of dead mites on pollen Lavandula offi cinalis Linalool 16.3 drawers in each treatment in each season are Linalyl acetate 13.0 presented in Table 3. (E)-anethole 75.3 According to the statistical data, all essential oils Foeniculum vulgare Limonene 8.7 were effi cient, but L. offi cinalis and thymol were Carvone 6.1 similarly and signifi cantly more effi cient against 1,8-cineole (eucalyptol) 54.6 Varroa in the fi rst season. However, in the second and Laurus nobilis α-terpinyl acetate 8.9 third seasons only the control group was signifi cantly Sabinene 7.0 diff erent from the treatment groups. All essential oils were thus found to be similarly effi cient against *Relative percentage obtained from GC peak area. Varroa in those seasons. 556 F. KÜTÜKOĞLU, A. O. GİRİŞGİN, L. AYDIN Table 3. Average number of dead mites dropped on pollen drawers per hive for 42 days in each essential oil. Season Essential oil Average number of dead mites per hive Standard error of mean Lavandula offi cinalis 125.8a 6.7 Foeniculum vulgare 75.1b 5.1 1 Laurus nobilis 45.0c 3.4 Th ymol 115.0a 7.4 Control 14.2d 1.1 Lavandula offi cinalis 97.5a 5.7 Foeniculum vulgare 82.0a 5.5 2 Laurus nobilis 54.5a 4.3 Th ymol 105.5a 5.7 Control 10.1b 0.9 Lavandula offi cinalis 123.1a 6.9 Foeniculum vulgare 77.8a 5.0 3 Laurus nobilis 44.5a 3.9 Th ymol 110.7a 6.4 Control 15.6b 1.3 a, b, c, d: values with diff erent letters in each category are signifi cantly diff erent. Th ere were 2 subgroups of control and no Th e composition of the essential oils tested in diff erence was observed between the subgroups. this work was obtained with GC/MS and was in Th ey were therefore treated as one control group in accordance with those reported by Adams (12). the statistical analysis. Several factors can infl uence the yield and the chemical composition of essential oils. Th erefore, it Discussion is possible that diff erent compositions and diff erent percentages were obtained by diff erent researchers Botanical extracts obtained from diff erent plant (17). species have been shown to have a broad spectrum of acaricidal activity against varroosis and also against In temperate climates, the best time to treat other organisms such as insects, mites, bacteria, varroosis is late autumn, and treatment should be fungi, and nematodes (10,16,17). performed prior to the bees overwintering (19). Accordingly, 2 autumn treatments were performed Because of the possibility of resistance in this research to better determine the effi cacies of development of mites and harmful residues in bee the essential oils. products, usage of organic compounds has become widespread throughout the world. At present, mite Satisfactory results have been reported from resistance to essential oils used to control Varroa the application of homemade essential oils such as or hazardous residues (except in misuses) have not thymol, Baccharis fl abellata (Eastern baccharis), been detected (10). Th ere can be an increase in Minthostachys verticillata (peperina), Lavandula thymol residue levels in honey due to the Th ymovar® spp. (lavender), Tagetes minuta (wild chamomile), treatment. However, there is no maximum residual etc. (10,17,20,21). However, in some rare cases, only level for thymol and the average thymol residue partial mite toxicity has been observed. When applied level in the honey from honeybee colonies that were as fumigants, the eff ectiveness of essential oils against treated with Th ymovar® is below the taste threshold Varroa mites depends greatly on temperature, time of (18). the year, and colony strength (16,21). 557 Varroacidal effi cacies of essential oils extracted from Lavandula offi cinalis, Foeniculum vulgare, and Laurus nobilis in naturally infested honeybee (Apis mellifera L.) colonies Th e percentage of effi cacy and control of the mite Our results showed that use of these 3 essential presented in this work seems to be low in comparison oils and thymol has a moderate eff ect on varroosis. to those obtained from synthetic acaricides commonly However, they can be used as a supplementary used to control Varroa, but the results are still promising. treatment with other organic compounds instead of Although the development of miticides from natural the use of synthetic acaricides, particularly because substances, like essential oils and organic acids (22), is they do not represent a resistance or sanitary risk. not likely to replace the use of conventional products No distinct adverse eff ects are expected on broods or in the control of the mite, it can still reduce the use of adult bees when the products are used properly. these chemicals to control Varroa. According to Mondet et al., treatment using thymol as an active substance can have some adverse eff ects Acknowledgments on forager bees, who initially appeared to be repelled Th is work was supported by the Commission of by the treatment but can become habituated to it (23). Scientifi c Research Projects of Uludağ University, Th is was supported in our work, where striking eff ects Bursa (project number: V–2006/28). We would were observed in the use of essential oils and thymol like to thank beekeeper Sebahattin Yılmaz for with forager bees being repelled in the fi rst minutes of permission to use his hives and the Department treatment. Similar results were observed in previous of Pharmacognosy, Faculty of Pharmacy, Anadolu research based on using the gel formulation of thymol University, Eskişehir, Turkey, for help in the analysis in the treatment of varroosis (24). of the essential oils. References 1. Genç, F., Aksoy, A.: Th e eff ects of infestation levels of Varroa 9. 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