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DOI 10.1007/s00436-010-2043-3
ORIGINAL PAPER
Received: 10 August 2010 / Accepted: 26 August 2010 / Published online: 14 September 2010
# Springer-Verlag 2010
Abstract Extracts of indigenous plants from South like promising natural compound to be incorporated for
America have shown a broad spectrum of bioactivities. the control of varroosis.
No-contaminant and natural substances have recently
resurged as control treatment options for varroosis in
honey bee colonies from Argentina. The aim of this Introduction
work was to evaluate the biological activity of botanical
extracts from Baccharis flabellata and Minthostachys Numerous plant-derived substances have demonstrated
verticillata on Varroa destructor and Apis mellifera. The physiological and behavioral activity against insect pests,
acaricidal and insecticidal activities were assessed by the and they can provide new sources for the development of
spraying application method. Both ethanolic extracts natural pesticides (George et al. 2008; Isman 2006).
showed high levels of toxicity against the mites and were Products with botanical origin have shown a wide range
harmless to their host, A. mellifera. During the attractive- of biological activities including toxicity, repellence, anti-
repellent test, the olfactory stimulus evoked for the extract feedant, and growth regulatory properties (Aivazi and
from B. flabellata resulted as a repellent for mites. The Vijayan 2009; Banchio et al. 2003, 2005; Ciccia et al.
aromatic stimulus of these extracts would be strong 2000; Ferrero et al. 2006; Jbilou et al. 2006). Extracts from
enough to cause disturbance on the behavior of V. indigenous plants from South America have shown a broad
destructor. Thus, the repellent effect of these substances spectrum antibacterial (Oliveira et al. 2007) and insecticidal
plus the toxicity on mites postulate these botanical extracts activity (Maggi et al. 2005; Palacios et al. 2007; Sosa and
Tonn 2008).
The genus Baccharis is represented by more than 500
N. Damiani (*) : L. B. Gende : M. D. Maggi : J. A. Marcangeli :
species, distributed mainly in Brazil, Argentina, Colom-
M. J. Eguaras
Laboratorio de Artrópodos. Facultad de Ciencias Exactas y bia, Chile, and Mexico (Giuliano 2001). The physico-
Naturales, Universidad Nacional de Mar del Plata, chemical aspects of the genus have been extensively
Funes 3350 (7600) Mar del Plata, studied since the early 1900s. More than 150 constituents
Buenos Aires, Argentina
have been isolated and identified. The main compounds
e-mail: ndamiani@mdp.edu.ar
include diterpenoids such as neo-clerodane, although
N. Damiani : L. B. Gende : M. D. Maggi : S. Palacios : biologically active components like derivates of kauranes
M. J. Eguaras and labdanes, phenols and essential oils have been
Consejo Nacional de Investigaciones Científicas y Técnicas
registered (Abad Martinez et al. 2005). Flavones, flavo-
(CONICET),
Capital Federal, Buenos Aires, Argentina noid glycosides, and coumarins and their derivates are the
phenolic classes with pharmacological proprieties present
S. Palacios in this species (Zdero et al. 1986). The genus has
Laboratorio de Química Fina y Productos Naturales,
demonstrated to possess a broad spectrum of antioxidant,
Universidad Católica de Córdoba,
Camino a Alta Gracia Km 10 (5000), antimicrobial, antifungal, and antiparasitic activity (Sarkar
Córdoba, Argentina et al. 2008; Verdi et al. 2005).
80 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
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 et al. 2009). Also, it has been proven as an effective defense
suggest that it would be necessary to define all phytochem- chemical because it interferes with feeding behavior,
ical constituents of botanical extracts in order to ensure development, and reproduction of the worm Spodoptera
reliability and repeatability of the investigation about their eridania (Gunderson et al. 1985). Sánchez-Ramos and
bioactivities (He 2000); however, most researchers con- Castañera (2000) demonstrated that seven natural mono-
clude that due to more than 150 chemical components terpenes (pulegone, eucalyptol, linalool, fenchone, men-
constituting an individual botanical extract (Cutler and thone, α-terpinene and γ-terpinene) showed high acaricidal
Cutler 1999), the full extract has to be considered as an activity against mobile stages of Tyrophagus putrescentiae.
active “compound”. Nevertheless, there are still debates Four flavones (jaceosidin, cirsiliol, nepetin/eupatorin,
about chromatographic and spectroscopic techniques that it hispidulin), seven neo-clerodane diterpenoids, oleanolic
has to be used in the characterization of the phytochemical acid, and a triterpene were isolated from B. flabellata
components of the extracts. Therefore, achieving a standard (Juan Hikawczuk et al. 2002; Saad et al. 1988; Verdi et al.
characterization technique is a future goal (He 2000; Ong 2005). Flavones are compounds derivative from benzo-γ-
2004). pyrone that belong to the flavonoid group (Harborne
Bioactivity of botanical extracts obtained from M. 1980). Certain species of Baccharis have flavones with
verticillata and B. flabellata was tested on V. destructor anti-inflammatory and antifungical proprieties (Gianello et
and A. mellifera at the present work. Minthostachys al. 1999; Rahalison et al. 1995). The essential oil from B.
verticillata is rich in essential oils conformed by (4R)(+)- flabellata was effective against Staphylococcus aureus
pulegone (69.70%), menthone (12.17%), trans-iso- (Derno et al. 2005). Marcucci (1995) registered flavones
pulegone (3.58%), and limonene (2.75%) as principal as abundant components in samples of bee propolis.
components (Palacios et al. 2009) together with menthol, Clerodane diterpenoids and triterpenes are natural prod-
isomenthone, piperitenone, sabinene, (E)-β-ocimene, 1,8- ucts derived from mevalonic acid widely distributed in
cineole, carvone, β-pinene, γ-pinene, myrcene in small nature (plants, fungi, insects, and other organisms), and
amounts. Besides the essential oils, little is known about the present structural variability and broad spectrum of
other chemical components of Minthostachys plants biological activity. Certain clerodane diterpenoids isolated
(Schmidt-Lebuhn 2008a). The insecticidal action of pule- from the genus Baccharis showed antifeedant and repel-
gone against Musca domestica has been reported (Palacios 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
Table 2 Number of mites on each section (E or C) after 90 min of antifeedant activity on Tribolium castaneum (Juan
exposition to different aromatic stimuli in the arenas for the attractive-
repellence test
Hikawczuk et al. 2006).
Recently, substances derived from plants, such as
a
E C Total P propolis extracts, some essential oils and their main
components, have resurged as a natural alternative for
M. verticillata 6 (0.3) 14 (0.7) 20 0.115
Varroa control in Argentina (Damiani et al. 2009, 2010a, b;
B. flabellata 5 (0.25) 15 (0.75) 20 0.041
Ruffinengo et al. 2005) where mites resistant to synthetic
Control 10 (0.5) 10 (0.5) 20 1
molecules have become a serious problem (Maggi et al.
Mites in the control arenas did not received differential aromatic 2009). Researches about the effects of botanical extracts on
stimulus. Brackets show the value in proportion honeybees’ diseases such as the American foulbrood have
a
Based in Z approximation showed promising results (Beoletto et al. 2008; Gende et al.
Parasitol Res (2011) 108:79–86 83
2008; González et al. 2007, 2008), but inconsistent issues (Damiani et al. 2010a). Some essential oils affected mite
have been found about the acaricidal or sublethal effects on reproduction and had a repellent action on the Varroa mite
V. destructor (Gende et al. 2005; Melathopoulos et al. during laboratory evaluations (Imdorf et al. 1999). Colin et
2000). Zaitoon (2001) showed that acetonic extracts from al. (1999) hypothesized that long-term repellency may
Rhazya stricta, Heliotropium bacciferum, and A. indica had reduce female mite fecundity. Very low concentrations of
remarkable in vitro toxicity against Varroa mites; however, monoterpenes or phenolic compounds could induce a
no toxic effects were registered when extract from A. indica reduction of the fecundity of the mites (Ellis and Baxendale
was orally supplied on infested bees neither when this 1997), thus preventing high levels of parasitism in bee
extract was sprayed on V. destructor and A. mellifera colonies. In our work, the olfactory stimulus evoked for the
(Melathopoulos et al. 2000); only pupae treated with the botanical extract from B. flabellata resulted as a repellent
extract resulted as a repellent for the mite (González- for V. destructor. There were no registered attractive or
Gómez et al. 2006). repellent effects on mites exposed to the M. verticillata
Lindberg et al. (2000) suggested that laboratory experi- extract, but it tends to be a repellent for mites, although no
ments must test mite and bee mortality simultaneously and significance statistically; however, the essential oil of M.
must allow us to investigate the effects of dose, exposure verticillata showed repellent properties against V. destruc-
time, and mode of application. In the present research, tor in laboratory tests (Ruffinengo et al. 2005) though its
infested bees were exposed to diverse concentrations of two toxicity was lower than other essential oils tested in the
different botanical alcoholic extracts applied topically by same study. These results suggest that the aromatic stimulus
spraying. Ethanolic extracts from M. verticillata and B. of these extracts would be strong enough to cause a
flabellata showed high levels of toxicity against the disturbance on the behavior of V. destructor. Thus, the
ectoparasitic mite V. destructor and were harmless to its repellent effect of these substances plus the toxicity
host, A. mellifera. Increasing the concentration and expo- observed on mites postulate these botanical extracts like a
sure time led to increase the toxicity of both botanical promising natural compound to be incorporated for the
extracts against Varroa mites. This biological activity can control of varroosis.
be attributed to different terpenes and phenolic compounds The concept of “Green Pesticides” refers to all types
that constitute these extracts (Domingo and López-Brea of nature-oriented and beneficial pest control materials
2003) as it has been demonstrated for other substances of that can contribute to reduce the pest population and
botanical origin with similar molecules in their chemical increase food production (Koul et al. 2008). They are
composition such as thyme and clove essential oils safe and eco-friendly. They are more compatible with the
(Damiani et al. 2009; Maggi et al. 2010). environmental components than synthetic pesticides
One of the main criticisms of using plant-derived (Isman and Machial 2006). Therefore, in recent years
treatments for Varroa control is that they have a narrow there is a return to the use of plants as a source of safer
range of mite selective doses (Kraus et al. 1994). A high pesticides to the environment and human health (Mansaray
selectivity index involves minor risks of adverse effects on 2000; Ottaway 2001). No doubt, natural insecticides from
bees. In our research, after just 24 h of exposure, the extracts of plants are a very interesting alternative to pest
selectivity ratios for both extracts were similar or higher control; in addition, only a low number of plants have been
than the ratios reported for essential oils with good efficacy evaluated in relation to the natural source available world-
against the mite (Damiani et al. 2009; Lindberg et al. 2000; wide, so there are important incentives for future researches.
Ruffinengo et al. 2005). Thus, both botanical extracts Argentina has a very rich natural floral diversity with
caused mite mortality without severe harmful effects on numerous medicinal and pharmacological properties that
adult honeybees. should be explored in depth (Goleniowski et al. 2006). In the
Besides the toxic effects, sublethal effects can be useful area of honeybee health, there is an unexplored way for
in controlling Varroa. Any substance that interferes in the future research involving alternative natural substances to
mite’s ability to locate its host may have a practical value as control this pest. Reduction of V. destructor population in
a method of control (Colin et al. 1994). Some natural honeybee colonies involves treatments with acceptable
substances used to reduce Varroa populations can evoke acaricidal activity with no side effects on honeybees that
physiological or behavioral modification on individual minimize residues in honey and wax and that constitute a
mite. Thymol exhibits high miticidal activity (Imdorf et viable alternative to reduce the mite resistance to synthetic
al. 1999) although superseding the queens in field con- acaricides.
ditions have been reported (Sammataro et al. 1998). After
topical treatment with propolis extracts, the mites remained Acknowledgements This study was supported by PICT REDES
in an inactive state of narcosis during the first hours of Project No. 00890 (ANPCYT) and Exa Project No. 456/09 (UNMDP)
treatments and regained their normal activity after that to ME, Argentina.
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