Parasitol Res (2011) 108:79–86
DOI 10.1007/s00436-010-2043-3
ORIGINAL PAPER
Repellent and acaricidal effects of botanical extracts
on 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 / Published online: 14 September 2010
# Springer-Verlag 2010
Abstract Extracts of indigenous plants from South
America have shown a broad spectrum of bioactivities.
No-contaminant and natural substances have recently
resurged as control treatment options for varroosis in
honey bee colonies from Argentina. The aim of this
work was to evaluate the biological activity of botanical
extracts from Baccharis flabellata and Minthostachys
verticillata on Varroa destructor and Apis mellifera. The
acaricidal and insecticidal activities were assessed by the
spraying application method. Both ethanolic extracts
showed high levels of toxicity against the mites and were
harmless to their host, A. mellifera. During the attractive-
repellent test, the olfactory stimulus evoked for the extract
from B. flabellata resulted as a repellent for mites. The
aromatic stimulus of these extracts would be strong
enough to cause disturbance on the behavior of V.
destructor. Thus, the repellent effect of these substances
plus the toxicity on mites postulate these botanical extracts
N. Damiani (*) : L. B. Gende : M. D. Maggi : J. A. Marcangeli :
M. J. Eguaras
Laboratorio de Artrópodos. Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Mar del Plata,
Funes 3350 (7600) Mar del Plata,
Buenos Aires, Argentina
e-mail: ndamiani@mdp.edu.ar
N. Damiani : L. B. Gende : M. D. Maggi : S. Palacios :
M. J. Eguaras
Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET),
Capital Federal, Buenos Aires, Argentina
S. Palacios
Laboratorio de Química Fina y Productos Naturales,
Universidad Católica de Córdoba,
Camino a Alta Gracia Km 10 (5000),
Córdoba, Argentina
like promising natural compound to be incorporated for
the control of varroosis.
Introduction
Numerous plant-derived substances have demonstrated
physiological and behavioral activity against insect pests,
and they can provide new sources for the development of
natural pesticides (George et al. 2008; Isman 2006).
Products with botanical origin have shown a wide range
of biological activities including toxicity, repellence, anti-
feedant, and growth regulatory properties (Aivazi and
Vijayan 2009; Banchio et al. 2003, 2005; Ciccia et al.
2000; Ferrero et al. 2006; Jbilou et al. 2006). Extracts from
indigenous plants from South America have shown a broad
spectrum antibacterial (Oliveira et al. 2007) and insecticidal
activity (Maggi et al. 2005; Palacios et al. 2007; Sosa and
Tonn 2008).
The genus Baccharis is represented by more than 500
species, distributed mainly in Brazil, Argentina, Colom-
bia, Chile, and Mexico (Giuliano 2001). The physico-
chemical aspects of the genus have been extensively
studied since the early 1900s. More than 150 constituents
have been isolated and identified. The main compounds
include diterpenoids such as neo-clerodane, although
biologically active components like derivates of kauranes
and labdanes, phenols and essential oils have been
registered (Abad Martinez et al. 2005). Flavones, flavo-
noid glycosides, and coumarins and their derivates are the
phenolic classes with pharmacological proprieties present
in this species (Zdero et al. 1986). The genus has
demonstrated to possess a broad spectrum of antioxidant,
antimicrobial, antifungal, and antiparasitic activity (Sarkar
et al. 2008; Verdi et al. 2005).
80
The genus Minthostachys is constituted by 17 species
restricted to the Andean region in South America (Schmidt-
Lebuhn 2008b). Different varieties of Minthostachys
verticillata have fundamentally ethnobotanical, pharmaco-
logical, and commercial interest. Their basic chemical
constituents including pulegone, menthone, menthol, iso-
menthone, piperine acid, 1,8-cineole, carvone, β-pinene, γ-
pinene, among others. Most studies are related to its
essential oil that showed some monoterpenes like pulegone,
menthone, limonene, and myrcene with antifungal (Alkire
et 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 a
parasitic mite, which is considered a severe pest for
honeybees causing serious losses to the beekeeper (de
Jong et al. 1982). The mite population levels in beehives
must be maintained below economic injury (Delaplane
and Hood 1997). Apis mellifera colonies are convention-
ally treated with synthetic acaricides. No-contaminant and
natural substances have recently resurged as control
treatment 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 carried
out using botanical extracts to varroosis control and the
results have not been conclusive. Only one research
showed that acetonic extracts of three plants had remark-
able toxicity against Varroa mite in laboratory conditions
(Zaitoon 2001). In Syria, a group of researchers observed
high mite mortality when beehives were fumigated with
extracts of seeds of Pimpinella anisum (Daher-Hjaij and
Alburaki 2006). In vitro assays evaluating the extract of
fruits of Melia azederach on mites and honeybees showed
low levels of bioactivity (Gende et al. 2005, 2008). No
toxic effects were registered when extract from Azadir-
achta indica was orally administered on infested bees
neither when this extract was sprayed on V. destructor and
A. mellifera (Melathopoulos et al. 2000); only pupae
treated with the extract resulted as a repellent for the mite
(González-Gómez et al. 2006).The repellent or attractive
effects, reproductive inhibition, narcosis, or any behav-
ioral disturbance have been the most common sublethal
effects reported in natural products tested against this
pest. A substance able to modify the feeding behavior,
development or reproduction of the mite inside the bee
colony can be useful in controlling Varroa. Any effect
that interferes in the mite’s ability to locate its host may
have a practical value as a method of control (Colin et al.
1994).
The aim of this work was to evaluate the biological
activity of botanical extracts from Baccharis flabellata and
M. verticillata on V. destructor and A. mellifera.
Parasitol Res (2011) 108:79–86
Materials and methods
Botanical ethanolic extracts
Plants were collected in Traslasierra Valley, Córdoba,
Argentina in 2007. A voucher specimen has been deposited
at the Herbarium Marcelino Sayago of the Faculty of
Agricultural Science, Universidad Católica de Córdoba and
was identified by the agronomist, Gustavo Ruiz.
The vegetable material was air-dried at room tempera-
ture, crushed, and extracted by 48 h of maceration with
ethanol. Viscous extracts were obtained after solvent
removal at reduced pressure.
The botanical extracts selected were obtained from aerial
parts of B. flabellata (Hook. & Arn.) (Asteraceae) and M.
verticillata (Griseb.) Epling (Lamiaceae).
Experimental animals
Apis mellifera colonies highly infested with V. destructor
were used. The hives were placed in an experimental apiary
of the National University of Mar del Plata, near Mar del
Plata, Argentina (38º10′06″ S; 57º38′10″ W). All colonies
had been left untreated for Varroa for the preceding 12–
24 months.
Adult female mites were collected from capped brood by
opening and inspecting individual cells. In order to avoid
starvation, the mites were kept on bee larvae or pupae in
cages during the collection process. Mites which seemed
newly moulted, weak, and abnormal were discarded since
they may have a different response during the bioassays.
Adult worker bees walking on brood combs were picked
up from colonies; they were inspected for Varroa before
being placed in the dishes for the tests.
Acaricidal and insecticidal activity bioassays
The viscous extracts were dissolved in 55% ethanol in
order to obtain the solutions of botanical extracts from
B. flabellata and M. verticillata. The concentrations used
in the treatment were 0.5%, 1%, 2%, 4%, 7%, and 10%
(w/v).
The spraying application method according to Damiani
et al. (2010b) was used. Petri dishes (90×15 mm) padded
with absorbent filter paper on the inner bottom and with an
extra lid of metallic mesh were used. Five adult female
mites and five adult worker bees (free of mites) were placed
in every modified Petri dish. Once the mites were attached
at the body of the bees in each experimental unit, 1 ml of
each concentration (for both botanical extracts individually)
was sprayed on the bees throughout the metallic lid using a
hand sprayer. A device with candy and water was placed
inside each unit as food for the bees. Five bees and five
Parasitol Res (2011) 108:79–86
mites in a modified Petri dish sprayed with alcohol 55%
were included as controls. Five replicates for each
experimental group were run. Bioassay dishes were placed
in incubators at 28±1°C and 60% RH. Death of bees and
mites was assessed at 24 and 48 h. Mortality was evaluated
by gently prodding each mite with a narrow paintbrush;
lack of response to consecutive stimulus over 1 min was
considered an indication of death. All bees (dead or
survivor bees) were visually inspected for the presence of
mites.
Statistical analyses were performed with a specific
software for the calculation of LC50 values (lethal
concentration that kills 50% of the exposed animals),
and 95% inverse confidence limits established by USEPA
(1986) and EPA software (version 1.5) according to
Lindberg 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 by
means of the greater LC50/lower LC50 ratio based on the
statistical value recommended in standard methods
(APHA 1992). Ratios were considered to be meaningful
indicators of selectivity only if the 95% confidence limits
of the LC50 values being compared did not overlap. A
selectivity ratio, calculated as A. mellifera LC 50 /V.
destructor LC50 was estimated for each treatment at each
observation 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 opposite
ends of the dish was placed a circle of paper filter
(diameter, 1 cm), one of which was embedded with 8 μl
of 10% botanical extract (on section E) and the other, only
with 55% alcoholic solution (on section C). Both solutions
applied on the circles were allowed to evaporate before
testing. At the beginning of the test, a single adult female
mite was placed on the central area of the arena. Twenty
observation arenas for each botanical extract were run
simultaneously. Devices without extract at both ends were
included as controls. The tests were made at room
temperature and darkness. After 90 min, mite location
according to the areas of division of each arena was
registered. As stated by Kraus et al. (1994), more
pronounced orientation effects can be observed during this
period of time.
The attractive-repellent effects of the botanical extracts
on the mites were analyzed by contrast to the observed
frequency of mites on each zone by means of a binomial
test for a two-level categorical dependent variable using an
SPSS software, version 11.5 for Windows.
81
Results
Effects of spraying applications on mites and bees
The estimated LC50 values obtained at each observation
time and selectivity ratios for both treatments are shown in
Table 1. LC50 values for mites treated with ethanolic
extracts of M. verticillata and B. flabellata were not
statistically different between them throughout both obser-
vation times. No mortality of bees was observed during
treatment even at the highest concentrations and 48 h after
treatment. The extract obtained from B. flabellata showed
the best selectivity index during both observation times.
Attractive and repellent effects
The number and proportion of mites found on different
sections of the arena after testing are showed in Table 2.
The binomial statistical analysis of mite location inside
the arena after testing with B. flabellata extract showed that
the observed proportion of mites on both zones of the
device was different to the expected observation (p<0.05);
however, the group of mites exposed to the olfactory
stimulus of M. verticillata behaved statistically similar to
the 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 used
for 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 of
parasites (Semmler et al. 2009) and pests (George et al.
2008; Isman 2006) with importance in agricultural and
veterinary industries. Botanical extracts obtained from
different plant species have showed a broad spectrum of
biological activity (Aivazi and Vijayan 2009; Banchio et al.
2003, 2005; Ciccia et al. 2000; Ferrero et al. 2006; Jbilou et
al. 2006). Each individual extract comprises a complex
unique mixture of different phytochemicals (plant second-
ary metabolites). The chemical nature of these constituents
varies considerably between species. The same herbal
extract may vary depending on the harvest season, plant
origin, drying process, and other factors. Some “character-
ized herbal extracts” commercially available for medical
use have specified one or two chemical constituents. This
determination, however, does not give a complete profile of
a botanical product because multiple constituents are
usually responsible for its therapeutic effects. The different
components of an extract could work synergistically and
82 Parasitol Res (2011) 108:79–86

Table 1 LC50 values+95% confidence limits and selectivity ratios estimated for each botanical extract against V. destructor and A. mellifera at 24
and 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 multiple
comparisons among all LC50 values, statistically significant differences were observed when the statistical value was greater than the
corresponding critical value set forth by APHA (1992). Lowercase letters compare between different observation times within each botanical
extract. Uppercase letters compare between both extracts within each observation time

should not be separated into active parts. Some authors
suggest that it would be necessary to define all phytochem-
ical constituents of botanical extracts in order to ensure
reliability and repeatability of the investigation about their
bioactivities (He 2000); however, most researchers con-
clude that due to more than 150 chemical components
constituting an individual botanical extract (Cutler and
Cutler 1999), the full extract has to be considered as an
active “compound”. Nevertheless, there are still debates
about chromatographic and spectroscopic techniques that it
has to be used in the characterization of the phytochemical
components of the extracts. Therefore, achieving a standard
characterization technique is a future goal (He 2000; Ong
2004).
Bioactivity of botanical extracts obtained from M.
verticillata and B. flabellata was tested on V. destructor
and A. mellifera at the present work. Minthostachys
verticillata is rich in essential oils conformed by (4R)(+)-
pulegone (69.70%), menthone (12.17%), trans-iso-
pulegone (3.58%), and limonene (2.75%) as principal
components (Palacios et al. 2009) together with menthol,
isomenthone, piperitenone, sabinene, (E)-β-ocimene, 1,8-
cineole, carvone, β-pinene, γ-pinene, myrcene in small
amounts. Besides the essential oils, little is known about the
other chemical components of Minthostachys plants
(Schmidt-Lebuhn 2008a). The insecticidal action of pule-
gone against Musca domestica has been reported (Palacios
Table 2 Number of mites on each section (E or C) after 90 min of
exposition to different aromatic stimuli in the arenas for the attractive-
repellence test
E C Total
M. verticillata 6 (0.3) 14 (0.7) 20
B. flabellata 5 (0.25) 15 (0.75) 20
Control 10 (0.5) 10 (0.5) 20
Mites in the control arenas did not received differential aromatic
stimulus. Brackets show the value in proportion
a
Based in Z approximation
et al. 2009). Also, it has been proven as an effective defense
chemical because it interferes with feeding behavior,
development, and reproduction of the worm Spodoptera
eridania (Gunderson et al. 1985). Sánchez-Ramos and
Castañera (2000) demonstrated that seven natural mono-
terpenes (pulegone, eucalyptol, linalool, fenchone, men-
thone, α-terpinene and γ-terpinene) showed high acaricidal
activity against mobile stages of Tyrophagus putrescentiae.
Four flavones (jaceosidin, cirsiliol, nepetin/eupatorin,
hispidulin), seven neo-clerodane diterpenoids, oleanolic
acid, 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 (Harborne
1980). Certain species of Baccharis have flavones with
anti-inflammatory and antifungical proprieties (Gianello et
al. 1999; Rahalison et al. 1995). The essential oil from B.
flabellata was effective against Staphylococcus aureus
(Derno et al. 2005). Marcucci (1995) registered flavones
as abundant components in samples of bee propolis.
Clerodane diterpenoids and triterpenes are natural prod-
ucts derived from mevalonic acid widely distributed in
nature (plants, fungi, insects, and other organisms), and
present structural variability and broad spectrum of
biological activity. Certain clerodane diterpenoids isolated
from the genus Baccharis showed antifeedant and repel-
lent activity against larvae of Tenebrio molitor (Sosa et al.
1994). Neo-clerodane diterpenes isolated from acetone
extracts of the aerial parts of B. flabellata showed
antifeedant activity on Tribolium castaneum (Juan
Hikawczuk et al. 2006).
Recently, substances derived from plants, such as
a
P propolis extracts, some essential oils and their main
components, have resurged as a natural alternative for
0.115
Varroa control in Argentina (Damiani et al. 2009, 2010a, b;
0.041
Ruffinengo et al. 2005) where mites resistant to synthetic
1
molecules have become a serious problem (Maggi et al.
2009). Researches about the effects of botanical extracts on
honeybees’ diseases such as the American foulbrood have
showed promising results (Beoletto et al. 2008; Gende et al.
Parasitol Res (2011) 108:79–86
2008; González et al. 2007, 2008), but inconsistent issues
have been found about the acaricidal or sublethal effects on
V. destructor (Gende et al. 2005; Melathopoulos et al.
2000). Zaitoon (2001) showed that acetonic extracts from
Rhazya stricta, Heliotropium bacciferum, and A. indica had
remarkable in vitro toxicity against Varroa mites; however,
no toxic effects were registered when extract from A. indica
was orally supplied on infested bees neither when this
extract was sprayed on V. destructor and A. mellifera
(Melathopoulos et al. 2000); only pupae treated with the
extract 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 and
must allow us to investigate the effects of dose, exposure
time, and mode of application. In the present research,
infested bees were exposed to diverse concentrations of two
different botanical alcoholic extracts applied topically by
spraying. Ethanolic extracts from M. verticillata and B.
flabellata showed high levels of toxicity against the
ectoparasitic mite V. destructor and were harmless to its
host, A. mellifera. Increasing the concentration and expo-
sure time led to increase the toxicity of both botanical
extracts against Varroa mites. This biological activity can
be attributed to different terpenes and phenolic compounds
that constitute these extracts (Domingo and López-Brea
2003) as it has been demonstrated for other substances of
botanical origin with similar molecules in their chemical
composition such as thyme and clove essential oils
(Damiani et al. 2009; Maggi et al. 2010).
One of the main criticisms of using plant-derived
treatments for Varroa control is that they have a narrow
range of mite selective doses (Kraus et al. 1994). A high
selectivity index involves minor risks of adverse effects on
bees. In our research, after just 24 h of exposure, the
selectivity ratios for both extracts were similar or higher
than the ratios reported for essential oils with good efficacy
against the mite (Damiani et al. 2009; Lindberg et al. 2000;
Ruffinengo et al. 2005). Thus, both botanical extracts
caused mite mortality without severe harmful effects on
adult honeybees.
Besides the toxic effects, sublethal effects can be useful
in controlling Varroa. Any substance that interferes in the
mite’s ability to locate its host may have a practical value as
a method of control (Colin et al. 1994). Some natural
substances used to reduce Varroa populations can evoke
physiological or behavioral modification on individual
mite. Thymol exhibits high miticidal activity (Imdorf et
al. 1999) although superseding the queens in field con-
ditions have been reported (Sammataro et al. 1998). After
topical treatment with propolis extracts, the mites remained
in an inactive state of narcosis during the first hours of
treatments and regained their normal activity after that
83
(Damiani et al. 2010a). Some essential oils affected mite
reproduction and had a repellent action on the Varroa mite
during laboratory evaluations (Imdorf et al. 1999). Colin et
al. (1999) hypothesized that long-term repellency may
reduce female mite fecundity. Very low concentrations of
monoterpenes or phenolic compounds could induce a
reduction of the fecundity of the mites (Ellis and Baxendale
1997), thus preventing high levels of parasitism in bee
colonies. In our work, the olfactory stimulus evoked for the
botanical extract from B. flabellata resulted as a repellent
for V. destructor. There were no registered attractive or
repellent effects on mites exposed to the M. verticillata
extract, but it tends to be a repellent for mites, although no
significance statistically; however, the essential oil of M.
verticillata showed repellent properties against V. destruc-
tor in laboratory tests (Ruffinengo et al. 2005) though its
toxicity was lower than other essential oils tested in the
same study. These results suggest that the aromatic stimulus
of these extracts would be strong enough to cause a
disturbance on the behavior of V. destructor. Thus, the
repellent effect of these substances plus the toxicity
observed on mites postulate these botanical extracts like a
promising natural compound to be incorporated for the
control of varroosis.
The concept of “Green Pesticides” refers to all types
of nature-oriented and beneficial pest control materials
that can contribute to reduce the pest population and
increase food production (Koul et al. 2008). They are
safe and eco-friendly. They are more compatible with the
environmental components than synthetic pesticides
(Isman and Machial 2006). Therefore, in recent years
there is a return to the use of plants as a source of safer
pesticides to the environment and human health (Mansaray
2000; Ottaway 2001). No doubt, natural insecticides from
extracts of plants are a very interesting alternative to pest
control; in addition, only a low number of plants have been
evaluated 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 with
numerous medicinal and pharmacological properties that
should be explored in depth (Goleniowski et al. 2006). In the
area of honeybee health, there is an unexplored way for
future research involving alternative natural substances to
control this pest. Reduction of V. destructor population in
honeybee colonies involves treatments with acceptable
acaricidal activity with no side effects on honeybees that
minimize residues in honey and wax and that constitute a
viable alternative to reduce the mite resistance to synthetic
acaricides.
Acknowledgements This study was supported by PICT REDES
Project No. 00890 (ANPCYT) and Exa Project No. 456/09 (UNMDP)
to ME, Argentina.
84
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