ORIGINAL PAPER

Repellent and acaricidal effects of botanical extractson Varroa destructor

Natalia Damiani & Liesel B. Gende & Matías D. Maggi &Sara Palacios & Jorge A. Marcangeli & Martín J. Eguaras

Received: 10 August 2010 /Accepted: 26 August 2010# Springer-Verlag 2010

Abstract Extracts of indigenous plants from SouthAmerica have shown a broad spectrum of bioactivities.No-contaminant and natural substances have recentlyresurged as control treatment options for varroosis inhoney bee colonies from Argentina. The aim of thiswork was to evaluate the biological activity of botanicalextracts from Baccharis flabellata and Minthostachysverticillata on Varroa destructor and Apis mellifera. Theacaricidal and insecticidal activities were assessed by thespraying application method. Both ethanolic extractsshowed high levels of toxicity against the mites and wereharmless to their host, A. mellifera. During the attractive-repellent test, the olfactory stimulus evoked for the extractfrom B. flabellata resulted as a repellent for mites. Thearomatic stimulus of these extracts would be strongenough to cause disturbance on the behavior of V.destructor. Thus, the repellent effect of these substancesplus the toxicity on mites postulate these botanical extracts

like promising natural compound to be incorporated forthe control of varroosis.

Introduction

Numerous plant-derived substances have demonstratedphysiological and behavioral activity against insect pests,and they can provide new sources for the development ofnatural pesticides (George et al. 2008; Isman 2006).Products with botanical origin have shown a wide rangeof biological activities including toxicity, repellence, anti-feedant, and growth regulatory properties (Aivazi andVijayan 2009; Banchio et al. 2003, 2005; Ciccia et al.2000; Ferrero et al. 2006; Jbilou et al. 2006). Extracts fromindigenous plants from South America have shown a broadspectrum antibacterial (Oliveira et al. 2007) and insecticidalactivity (Maggi et al. 2005; Palacios et al. 2007; Sosa andTonn 2008).

The genus Baccharis is represented by more than 500species, distributed mainly in Brazil, Argentina, Colom-bia, Chile, and Mexico (Giuliano 2001). The physico-chemical aspects of the genus have been extensivelystudied since the early 1900s. More than 150 constituentshave been isolated and identified. The main compoundsinclude diterpenoids such as neo-clerodane, althoughbiologically active components like derivates of kauranesand labdanes, phenols and essential oils have beenregistered (Abad Martinez et al. 2005). Flavones, flavo-noid glycosides, and coumarins and their derivates are thephenolic classes with pharmacological proprieties presentin this species (Zdero et al. 1986). The genus hasdemonstrated to possess a broad spectrum of antioxidant,antimicrobial, antifungal, and antiparasitic activity (Sarkaret al. 2008; Verdi et al. 2005).

N. Damiani (*) : L. B. Gende :M. D. Maggi : J. A. Marcangeli :M. J. EguarasLaboratorio de Artrópodos. Facultad de Ciencias Exactas yNaturales, Universidad Nacional de Mar del Plata,Funes 3350 (7600) Mar del Plata,Buenos Aires, Argentinae-mail: ndamiani@mdp.edu.ar

N. Damiani : L. B. Gende :M. D. Maggi : S. Palacios :M. J. EguarasConsejo Nacional de Investigaciones Científicas y Técnicas(CONICET),Capital Federal, Buenos Aires, Argentina

S. PalaciosLaboratorio de Química Fina y Productos Naturales,Universidad Católica de Córdoba,Camino a Alta Gracia Km 10 (5000),Córdoba, Argentina

Parasitol ResDOI 10.1007/s00436-010-2043-3

The genus Minthostachys is constituted by 17 speciesrestricted to the Andean region in South America (Schmidt-Lebuhn 2008b). Different varieties of Minthostachysverticillata have fundamentally ethnobotanical, pharmaco-logical, and commercial interest. Their basic chemicalconstituents including pulegone, menthone, menthol, iso-menthone, piperine acid, 1,8-cineole, carvone, β-pinene, γ-pinene, among others. Most studies are related to itsessential oil that showed some monoterpenes like pulegone,menthone, limonene, and myrcene with antifungal (Alkireet al. 1994; Cano et al. 2008), antibacterial (Rojas et al.2003), antiparasitic (Schmidt-Lebuhn 2008a), and insecti-cidal activity (Palacios et al. 2009).

Varroa destructor (Anderson and Trueman 2000) is aparasitic mite, which is considered a severe pest forhoneybees causing serious losses to the beekeeper (deJong et al. 1982). The mite population levels in beehivesmust be maintained below economic injury (Delaplaneand Hood 1997). Apis mellifera colonies are convention-ally treated with synthetic acaricides. No-contaminant andnatural substances have recently resurged as controltreatment options in Argentina (Damiani et al. 2009,2010a; Ruffinengo et al. 2005) to minimize the develop-ment of acaricide resistance in V. destructor populations(Maggi et al. 2009). Very little studies have been carriedout using botanical extracts to varroosis control and theresults have not been conclusive. Only one researchshowed that acetonic extracts of three plants had remark-able toxicity against Varroa mite in laboratory conditions(Zaitoon 2001). In Syria, a group of researchers observedhigh mite mortality when beehives were fumigated withextracts of seeds of Pimpinella anisum (Daher-Hjaij andAlburaki 2006). In vitro assays evaluating the extract offruits of Melia azederach on mites and honeybees showedlow levels of bioactivity (Gende et al. 2005, 2008). Notoxic effects were registered when extract from Azadir-achta indica was orally administered on infested beesneither when this extract was sprayed on V. destructor andA. mellifera (Melathopoulos et al. 2000); only pupaetreated with the extract resulted as a repellent for the mite(González-Gómez et al. 2006).The repellent or attractiveeffects, reproductive inhibition, narcosis, or any behav-ioral disturbance have been the most common sublethaleffects reported in natural products tested against thispest. A substance able to modify the feeding behavior,development or reproduction of the mite inside the beecolony can be useful in controlling Varroa. Any effectthat interferes in the mite’s ability to locate its host mayhave a practical value as a method of control (Colin et al.1994).

The aim of this work was to evaluate the biologicalactivity of botanical extracts from Baccharis flabellata andM. verticillata on V. destructor and A. mellifera.

Materials and methods

Botanical ethanolic extracts

Plants were collected in Traslasierra Valley, Córdoba,Argentina in 2007. A voucher specimen has been depositedat the Herbarium Marcelino Sayago of the Faculty ofAgricultural Science, Universidad Católica de Córdoba andwas identified by the agronomist, Gustavo Ruiz.

The vegetable material was air-dried at room tempera-ture, crushed, and extracted by 48 h of maceration withethanol. Viscous extracts were obtained after solventremoval at reduced pressure.

The botanical extracts selected were obtained from aerialparts of B. flabellata (Hook. & Arn.) (Asteraceae) and M.verticillata (Griseb.) Epling (Lamiaceae).

Experimental animals

Apis mellifera colonies highly infested with V. destructorwere used. The hives were placed in an experimental apiaryof the National University of Mar del Plata, near Mar delPlata, Argentina (38º10′06″ S; 57º38′10″ W). All colonieshad been left untreated for Varroa for the preceding 12–24 months.

Adult female mites were collected from capped brood byopening and inspecting individual cells. In order to avoidstarvation, the mites were kept on bee larvae or pupae incages during the collection process. Mites which seemednewly moulted, weak, and abnormal were discarded sincethey may have a different response during the bioassays.

Adult worker bees walking on brood combs were pickedup from colonies; they were inspected for Varroa beforebeing placed in the dishes for the tests.

Acaricidal and insecticidal activity bioassays

The viscous extracts were dissolved in 55% ethanol inorder to obtain the solutions of botanical extracts fromB. flabellata and M. verticillata. The concentrations usedin the treatment were 0.5%, 1%, 2%, 4%, 7%, and 10%(w/v).

The spraying application method according to Damianiet al. (2010b) was used. Petri dishes (90×15 mm) paddedwith absorbent filter paper on the inner bottom and with anextra lid of metallic mesh were used. Five adult femalemites and five adult worker bees (free of mites) were placedin every modified Petri dish. Once the mites were attachedat the body of the bees in each experimental unit, 1 ml ofeach concentration (for both botanical extracts individually)was sprayed on the bees throughout the metallic lid using ahand sprayer. A device with candy and water was placedinside each unit as food for the bees. Five bees and five

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mites in a modified Petri dish sprayed with alcohol 55%were included as controls. Five replicates for eachexperimental group were run. Bioassay dishes were placedin incubators at 28±1°C and 60% RH. Death of bees andmites was assessed at 24 and 48 h. Mortality was evaluatedby gently prodding each mite with a narrow paintbrush;lack of response to consecutive stimulus over 1 min wasconsidered an indication of death. All bees (dead orsurvivor bees) were visually inspected for the presence ofmites.

Statistical analyses were performed with a specificsoftware for the calculation of LC50 values (lethalconcentration that kills 50% of the exposed animals),and 95% inverse confidence limits established by USEPA(1986) and EPA software (version 1.5) according toLindberg et al. (2000). Abbott’s correction (Abbott 1925)and the alternative method of Litchfield and Wilcoxon(1949) were included in the analyses. Multiple compar-isons were carried out among all LC50 values obtained bymeans of the greater LC50/lower LC50 ratio based on thestatistical value recommended in standard methods(APHA 1992). Ratios were considered to be meaningfulindicators of selectivity only if the 95% confidence limitsof the LC50 values being compared did not overlap. Aselectivity ratio, calculated as A. mellifera LC50/V.destructor LC50 was estimated for each treatment at eachobservation time.

Attractive and repellent test

The observation arenas for the attractive-repellence experi-ments were made using a disposable Petri dish (diameter,9 cm) divided in two sections (E and C). Over oppositeends of the dish was placed a circle of paper filter(diameter, 1 cm), one of which was embedded with 8 μlof 10% botanical extract (on section E) and the other, onlywith 55% alcoholic solution (on section C). Both solutionsapplied on the circles were allowed to evaporate beforetesting. At the beginning of the test, a single adult femalemite was placed on the central area of the arena. Twentyobservation arenas for each botanical extract were runsimultaneously. Devices without extract at both ends wereincluded as controls. The tests were made at roomtemperature and darkness. After 90 min, mite locationaccording to the areas of division of each arena wasregistered. As stated by Kraus et al. (1994), morepronounced orientation effects can be observed during thisperiod of time.

The attractive-repellent effects of the botanical extractson the mites were analyzed by contrast to the observedfrequency of mites on each zone by means of a binomialtest for a two-level categorical dependent variable using anSPSS software, version 11.5 for Windows.

Results

Effects of spraying applications on mites and bees

The estimated LC50 values obtained at each observationtime and selectivity ratios for both treatments are shown inTable 1. LC50 values for mites treated with ethanolicextracts of M. verticillata and B. flabellata were notstatistically different between them throughout both obser-vation times. No mortality of bees was observed duringtreatment even at the highest concentrations and 48 h aftertreatment. The extract obtained from B. flabellata showedthe best selectivity index during both observation times.

Attractive and repellent effects

The number and proportion of mites found on differentsections of the arena after testing are showed in Table 2.

The binomial statistical analysis of mite location insidethe arena after testing with B. flabellata extract showed thatthe observed proportion of mites on both zones of thedevice was different to the expected observation (p<0.05);however, the group of mites exposed to the olfactorystimulus of M. verticillata behaved statistically similar tothe control group that did not receive differential stimula-tion (p=0.115). Therefore, the botanical extract from B.flabellata showed a significant repellent effect on V.destructor (p=0.041). None of the botanical extracts usedfor the test showed signs of attractive effects.

Discussion

Nowadays, a lot of researchers tend to return to inves-tigations involving plant extracts for a natural control ofparasites (Semmler et al. 2009) and pests (George et al.2008; Isman 2006) with importance in agricultural andveterinary industries. Botanical extracts obtained fromdifferent plant species have showed a broad spectrum ofbiological activity (Aivazi and Vijayan 2009; Banchio et al.2003, 2005; Ciccia et al. 2000; Ferrero et al. 2006; Jbilou etal. 2006). Each individual extract comprises a complexunique mixture of different phytochemicals (plant second-ary metabolites). The chemical nature of these constituentsvaries considerably between species. The same herbalextract may vary depending on the harvest season, plantorigin, drying process, and other factors. Some “character-ized herbal extracts” commercially available for medicaluse have specified one or two chemical constituents. Thisdetermination, however, does not give a complete profile ofa botanical product because multiple constituents areusually responsible for its therapeutic effects. The differentcomponents of an extract could work synergistically and

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should not be separated into active parts. Some authorssuggest that it would be necessary to define all phytochem-ical constituents of botanical extracts in order to ensurereliability and repeatability of the investigation about theirbioactivities (He 2000); however, most researchers con-clude that due to more than 150 chemical componentsconstituting an individual botanical extract (Cutler andCutler 1999), the full extract has to be considered as anactive “compound”. Nevertheless, there are still debatesabout chromatographic and spectroscopic techniques that ithas to be used in the characterization of the phytochemicalcomponents of the extracts. Therefore, achieving a standardcharacterization technique is a future goal (He 2000; Ong2004).

Bioactivity of botanical extracts obtained from M.verticillata and B. flabellata was tested on V. destructorand A. mellifera at the present work. Minthostachysverticillata is rich in essential oils conformed by (4R)(+)-pulegone (69.70%), menthone (12.17%), trans-iso-pulegone (3.58%), and limonene (2.75%) as principalcomponents (Palacios et al. 2009) together with menthol,isomenthone, piperitenone, sabinene, (E)-β-ocimene, 1,8-cineole, carvone, β-pinene, γ-pinene, myrcene in smallamounts. Besides the essential oils, little is known about theother chemical components of Minthostachys plants(Schmidt-Lebuhn 2008a). The insecticidal action of pule-gone against Musca domestica has been reported (Palacios

et al. 2009). Also, it has been proven as an effective defensechemical because it interferes with feeding behavior,development, and reproduction of the worm Spodopteraeridania (Gunderson et al. 1985). Sánchez-Ramos andCastañera (2000) demonstrated that seven natural mono-terpenes (pulegone, eucalyptol, linalool, fenchone, men-thone, α-terpinene and γ-terpinene) showed high acaricidalactivity against mobile stages of Tyrophagus putrescentiae.

Four flavones (jaceosidin, cirsiliol, nepetin/eupatorin,hispidulin), seven neo-clerodane diterpenoids, oleanolicacid, and a triterpene were isolated from B. flabellata(Juan Hikawczuk et al. 2002; Saad et al. 1988; Verdi et al.2005). Flavones are compounds derivative from benzo-γ-pyrone that belong to the flavonoid group (Harborne1980). Certain species of Baccharis have flavones withanti-inflammatory and antifungical proprieties (Gianello etal. 1999; Rahalison et al. 1995). The essential oil from B.flabellata was effective against Staphylococcus aureus(Derno et al. 2005). Marcucci (1995) registered flavonesas abundant components in samples of bee propolis.Clerodane diterpenoids and triterpenes are natural prod-ucts derived from mevalonic acid widely distributed innature (plants, fungi, insects, and other organisms), andpresent structural variability and broad spectrum ofbiological activity. Certain clerodane diterpenoids isolatedfrom the genus Baccharis showed antifeedant and repel-lent activity against larvae of Tenebrio molitor (Sosa et al.1994). Neo-clerodane diterpenes isolated from acetoneextracts of the aerial parts of B. flabellata showedantifeedant activity on Tribolium castaneum (JuanHikawczuk et al. 2006).

Recently, substances derived from plants, such aspropolis extracts, some essential oils and their maincomponents, have resurged as a natural alternative forVarroa control in Argentina (Damiani et al. 2009, 2010a, b;Ruffinengo et al. 2005) where mites resistant to syntheticmolecules have become a serious problem (Maggi et al.2009). Researches about the effects of botanical extracts onhoneybees’ diseases such as the American foulbrood haveshowed promising results (Beoletto et al. 2008; Gende et al.

Table 1 LC50 values+95% confidence limits and selectivity ratios estimated for each botanical extract against V. destructor and A. mellifera at 24and 48 h after treatment by spraying

LC50 mites+(95% confidence limits) LC50 honeybees+(95% confidence limits) Selectivity ratio

24 h 48 h 24 h 48 h 24 h 48 h

M. verticillata 2.16 (1.32–2.98) Aa 1.44 (0.97–1.86) Aa >10 >10 >5.63 >6.94

B. flabellata 1.57 (1.08–2.02) Aa 1.14 (0.89–1.36) Aa >10 >10 >6.37 >8.77

LC50 values (lethal concentration that kills 50% of the exposure animals) are expressed in treatment concentration (%)/Petri dish. In the multiplecomparisons among all LC50 values, statistically significant differences were observed when the statistical value was greater than thecorresponding critical value set forth by APHA (1992). Lowercase letters compare between different observation times within each botanicalextract. Uppercase letters compare between both extracts within each observation time

Table 2 Number of mites on each section (E or C) after 90 min ofexposition to different aromatic stimuli in the arenas for the attractive-repellence test

E C Total P a

M. verticillata 6 (0.3) 14 (0.7) 20 0.115

B. flabellata 5 (0.25) 15 (0.75) 20 0.041

Control 10 (0.5) 10 (0.5) 20 1

Mites in the control arenas did not received differential aromaticstimulus. Brackets show the value in proportiona Based in Z approximation

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2008; González et al. 2007, 2008), but inconsistent issueshave been found about the acaricidal or sublethal effects onV. destructor (Gende et al. 2005; Melathopoulos et al.2000). Zaitoon (2001) showed that acetonic extracts fromRhazya stricta, Heliotropium bacciferum, and A. indica hadremarkable in vitro toxicity against Varroa mites; however,no toxic effects were registered when extract from A. indicawas orally supplied on infested bees neither when thisextract was sprayed on V. destructor and A. mellifera(Melathopoulos et al. 2000); only pupae treated with theextract resulted as a repellent for the mite (González-Gómez et al. 2006).

Lindberg et al. (2000) suggested that laboratory experi-ments must test mite and bee mortality simultaneously andmust allow us to investigate the effects of dose, exposuretime, and mode of application. In the present research,infested bees were exposed to diverse concentrations of twodifferent botanical alcoholic extracts applied topically byspraying. Ethanolic extracts from M. verticillata and B.flabellata showed high levels of toxicity against theectoparasitic mite V. destructor and were harmless to itshost, A. mellifera. Increasing the concentration and expo-sure time led to increase the toxicity of both botanicalextracts against Varroa mites. This biological activity canbe attributed to different terpenes and phenolic compoundsthat constitute these extracts (Domingo and López-Brea2003) as it has been demonstrated for other substances ofbotanical origin with similar molecules in their chemicalcomposition such as thyme and clove essential oils(Damiani et al. 2009; Maggi et al. 2010).

One of the main criticisms of using plant-derivedtreatments for Varroa control is that they have a narrowrange of mite selective doses (Kraus et al. 1994). A highselectivity index involves minor risks of adverse effects onbees. In our research, after just 24 h of exposure, theselectivity ratios for both extracts were similar or higherthan the ratios reported for essential oils with good efficacyagainst the mite (Damiani et al. 2009; Lindberg et al. 2000;Ruffinengo et al. 2005). Thus, both botanical extractscaused mite mortality without severe harmful effects onadult honeybees.

Besides the toxic effects, sublethal effects can be usefulin controlling Varroa. Any substance that interferes in themite’s ability to locate its host may have a practical value asa method of control (Colin et al. 1994). Some naturalsubstances used to reduce Varroa populations can evokephysiological or behavioral modification on individualmite. Thymol exhibits high miticidal activity (Imdorf etal. 1999) although superseding the queens in field con-ditions have been reported (Sammataro et al. 1998). Aftertopical treatment with propolis extracts, the mites remainedin an inactive state of narcosis during the first hours oftreatments and regained their normal activity after that

(Damiani et al. 2010a). Some essential oils affected mitereproduction and had a repellent action on the Varroa miteduring laboratory evaluations (Imdorf et al. 1999). Colin etal. (1999) hypothesized that long-term repellency mayreduce female mite fecundity. Very low concentrations ofmonoterpenes or phenolic compounds could induce areduction of the fecundity of the mites (Ellis and Baxendale1997), thus preventing high levels of parasitism in beecolonies. In our work, the olfactory stimulus evoked for thebotanical extract from B. flabellata resulted as a repellentfor V. destructor. There were no registered attractive orrepellent effects on mites exposed to the M. verticillataextract, but it tends to be a repellent for mites, although nosignificance statistically; however, the essential oil of M.verticillata showed repellent properties against V. destruc-tor in laboratory tests (Ruffinengo et al. 2005) though itstoxicity was lower than other essential oils tested in thesame study. These results suggest that the aromatic stimulusof these extracts would be strong enough to cause adisturbance on the behavior of V. destructor. Thus, therepellent effect of these substances plus the toxicityobserved on mites postulate these botanical extracts like apromising natural compound to be incorporated for thecontrol of varroosis.

The concept of “Green Pesticides” refers to all typesof nature-oriented and beneficial pest control materialsthat can contribute to reduce the pest population andincrease food production (Koul et al. 2008). They aresafe and eco-friendly. They are more compatible with theenvironmental components than synthetic pesticides(Isman and Machial 2006). Therefore, in recent yearsthere is a return to the use of plants as a source of saferpesticides to the environment and human health (Mansaray2000; Ottaway 2001). No doubt, natural insecticides fromextracts of plants are a very interesting alternative to pestcontrol; in addition, only a low number of plants have beenevaluated in relation to the natural source available world-wide, so there are important incentives for future researches.Argentina has a very rich natural floral diversity withnumerous medicinal and pharmacological properties thatshould be explored in depth (Goleniowski et al. 2006). In thearea of honeybee health, there is an unexplored way forfuture research involving alternative natural substances tocontrol this pest. Reduction of V. destructor population inhoneybee colonies involves treatments with acceptableacaricidal activity with no side effects on honeybees thatminimize residues in honey and wax and that constitute aviable alternative to reduce the mite resistance to syntheticacaricides.

Acknowledgements This study was supported by PICT REDESProject No. 00890 (ANPCYT) and Exa Project No. 456/09 (UNMDP)to ME, Argentina.

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