Parasitol Res (2010) 107:1189–1192
DOI 10.1007/s00436-010-1986-8
ORIGINAL PAPER
Resistance phenomena to amitraz from populations
of the ectoparasitic mite Varroa destructor of Argentina
Matías D. Maggi & Sergio R. Ruffinengo & Pedro Negri &
Martín J. Eguaras
Received: 28 May 2010 / Accepted: 13 July 2010 / Published online: 29 July 2010
# Springer-Verlag 2010
Abstract In Argentina, Varroa destructor resistance to
coumaphos has been previously reported. However, the
status of mite susceptibility to other hard acaricides is
still unknown. At present, high infestation levels of V.
destructor are being detected in colonies of Apis mellifera
after treatment with amitraz. The aim of the present study
was to determine the LC50 of amitraz in V. destructor from
three apiaries with high mite density after treatment with
the acaricide. The LC50 values were 3.9, 3.5, and 3.7 μg/
Petri dish for mites from three different apiaries. Signif-
icant LC50 differences were detected between resistant and
susceptible mites. LC50 increased 35–39-fold when com-
pared to the corresponding baseline, suggesting the
development of resistance. These results are the first
report of resistance to amitraz in V. destructor in Argentina
and extend the knowledge according to the status of
acaricides resistance in the country.
M. D. Maggi : P. Negri : M. J. Eguaras
Laboratorio de Artrópodos, Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Mar del Plata,
Funes, 3350,
7600 Mar del Plata, Argentina
M. D. Maggi (*) : M. J. Eguaras
Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET),
Rivadavia 1917,
C1033AJ Buenos Aires, Argentina
e-mail: mmaggi@conicet.gov.ar
S. R. Ruffinengo
Cátedra de Apicultura, Facultad de Ciencias Agrarias,
Universidad Nacional de Mar del Plata,
Ruta Nacional 226, Km. 73.5, CC 276,
(B7620ZAA) Balcarce, Argentina
Introduction
The ectoparasitic mite Varroa destructor Anderson and
Trueman (2000) appear as a serious problem for beekeeping
when it became capable to parasite Apis mellifera L. colonies
in the middle of 1950 (Eguaras and Ruffinengo 2006). Due
to its 40 years of persistence in honey bee apiaries, V.
destructor is considered as the major pest of honey bee A.
mellifera (Rosenkranz et al. 2010). The serious effect of this
parasite is due to the parasitism of both adult bees and brood
cells. Adult mated mite females settle on bee workers and
drones, enter the brood cells a few hours before sealing, and
reproduce in close synchronization with the bee development
(Maggi et al. 2010a). After piercing the bee cuticle, the mite
females suck the hemolymph, taking proteins and contribut-
ing at the same time to the inoculation and multiplication of
bee pathogens. The combination of both injuries results in
the death of the colonies (revised in Rosenkranz et al. 2010).
The beekeeping industry has limited products to control
these mites. Historically, tau-fluvalinate has been the
miticide of choice for V. destructor control. It has been
used widespread throughout many years resulting in a
strong selective pressure on mite populations. Therefore,
resistant mite populations have emerged in numerous
countries (Macedo et al. 2002; Milani 1995). In areas
where tau-fluvalinate resistance was prevalent, beekeepers
began to apply the organophosphate coumaphos as
alternative treatment method to confront this problem
(Elzen et al. 2000). However, resistance to this pesticide
has already been reported in Europe and the USA (Elzen
and Westervelt 2002). Amitraz (formamidine) has been
also used for V. destructor control worldwide, and mite
resistance to this acaricide was reported, too, although few
cases were reported (Dujin et al. 1991 as cited by Milani
1999; Elzen et al. 1999; Rodriguez-Dehaibes et al. 2005).
1190 Parasitol Res (2010) 107:1189–1192

Table 1 Comparative results of probit analysis from response
concentration–mortality of amitraz on Varroa destructor in Argentina
among the present data and those of Maggi et al. (2008)
Origin of LC50 (μg/ 95% Degrees Resistance
mites Petri dish) Confidence of index
interval freedom
Apiary I 3.9 a 0.2–7.6 3 39
Apiary II 3.5 a 2.1–5.7 3 35
Apiary III 3.7 a 0.4–6.2 3 37
Maggi et 0.1 b 3.25e−2–0.15 2 –
al. (2008)
Different letters within the column indicate significant differences
(Fisher’s exact test, P<0.05). Resistance index is equal to LC50
resistant mites divided by LC50 of susceptible mites
Since its detection in Argentina in 1976 (Montiel and
Piola 1976), several chemical treatments have been used to
control this ectoparasite, including coumaphos, fluvalinate,
flumethrin, and amitraz with specific formulation for
apicultural use. Around 1990, tau-fluvalinate application
was nearly 100% effective for control of Varroa mites in
Argentina. However, high infestation levels of V. destructor
were detected in 1996 in colonies of A. mellifera after
treatment with this pyrethroid (Fernandez and García
1997). As a consequence, beekeepers began applying
coumaphos and amitraz in various formulations (Eguaras
and Ruffinengo 2006). Due to the high infestation levels of
V. destructor that are continuously being detected in
colonies of A. mellifera after treatment with synthetic
acaricides in Argentina, several studies were conducted to
detect Varroa resistance episodes to hard miticides (Maggi
et al. 2008). By this mean, resistance to coumaphos in the
north of Argentina has been reported (Maggi et al. 2009),
and currently, new cases are being detected in other
locations from this country (Ruffinengo et al. 2010).
The objective of this research was to determine if
resistance episodes to amitraz was involved in V. destructor
populations sampled from three commercial apiaries where
high infestation levels of the mite were detected after
treatment with amitraz was applied.
Material and methods
In autumn 2010, three commercial beekeepers from Santa
Fe, Argentina, detected high V. destructor infestation
following treatment of their colonies with amitraz at their
corresponding apiaries. To determine if acaricide resistance
was involved, bioassays were conducted on mite populations
from Santa Fe and compared with data from susceptible mite
populations from Mar del Plata, Buenos Aires, Argentina
(Maggi et al. 2008). V. destructor mites were obtained from
different infested A. mellifera combs from each apiary. Mites
were taken from capped brood by opening and inspecting
individual cells. Mature mites were removed from bee
broods with a slender moistened paint brush, placed in an
incubation stove at 70% RH and 33–34°C on bee larvae, and
kept in a glass Petri dish for 1–3 h until the number of mites
collected was sufficient. LC50 for amitraz was determined
using a toxicity method (Maggi et al. 2008). Technical
grades of amitraz (Pestanal®, Sigma–Aldrich) were diluted
in 1 ml of hexane (Cicarelli Laboratory, Argentina, Pro-
analysis). Concentrations increased from 0 to 75 μg/ml were
applied to the bottom of the dish for the acaricide. Five
replicates were done for each concentration and control.
Calculations of LC50 values and 95% fiducial limits, as
established by USEPA (1986), were conducted using EPA
software (version 1.5) as recommended by Lindberg et al.
(2000). Mortality values were adjusted in accordance with
Abbott (1925) as a function of natural mortality. LC50 values
and resistance indexes carried out among data were
compared between the present study and Maggi et al.
(2008); resistance index was calculated as LC50 “resistant”
mites/LC50 susceptible mites and statistically analyzed with
the Fisher exact test (APHA 1992).
Results and discussion
For mites from all three apiaries, the amitraz LC50 increased
35–39-fold (Table 1), when compared to the corresponding
baseline, suggesting the development of resistance. LC50
differences were detected between resistant and susceptible

Table 2 History of use of acaricides in the apiaries parasitized by V. destructor in recent years

Year Apiary I Apiary II Apiary III

2007 Autumn: flumethrin Autumn: flumethrin Autumn: flumethrin

Spring: flumethrin Spring: flumethrin Spring: flumethrin
2008 Autumn: amitraz Autumn: amitraz Autumn: amitraz
Spring: amitraz Spring: amitraz Spring: amitraz
2009 Autumn: flumethrin Autumn: flumethrin Autumn: flumethrin
Spring: acid oxalic Spring: acid oxalic Spring: acid oxalic
2010 Autumn: amitraz (failures detected) Autumn: amitraz (failures detected) Autumn: amitraz (failures detected)
Parasitol Res (2010) 107:1189–1192
Fig. 1 Geographical locations from Argentina where V. destructor
populations resistant to coumaphos (Maggi et al. 2009; Ruffinengo et
al. 2010) and to amitraz (present study) were detected. 1 amitraz
resistance, present study (29°20′49.78″ S; 59°58′4.79″ W); 2
coumaphos resistance (Maggi et al. 2009; 30°45′26.22″ S 57°59′
29.89″ W); 3 coumaphos resistance (Ruffinengo et al. 2010; 37°9′
0.97″ S; 58°28′58.70″ W); 4 coumaphos resistance (Ruffinengo et al.
2010; 34°27′21.26″ S; 58°54′29.98″ W); and 5 coumaphos resistance
(Ruffinengo et al. 2010; 36°46′29.70″ S; 59°5′23.23″ O)
mites (P<0.05; Table 1). The acaricides used for the control
of V. destructor populations in the studied apiaries in the
last 3 years are detailed in Table 2. Figure 1 shows the
geographical locations from Argentina, where V. destructor
resistance phenomena to acaricide were detected.
The current study documents high levels of mite
resistance to amitraz in Santa Fe province and extends the
1191
knowledge of V. destructor resistance to acaricides in
Argentina. Argentine regulations (as stated by SENASA)
require one Amivar® strips (1 g amitraz per strip) or one
tablet ABVarA® (400 mg amitraz; beekeeper’s choice) to
be placed in every hive. Nevertheless, some beekeepers
also use homemade formulations based on amitraz. Taking
into account these results, in concordance with previous
reports of resistance to coumaphos in Argentina (Maggi et
al. 2009), control strategies must be improved. If not,
resistance may develop and spread rapidly with serious
economic losses from Argentinean beekeepers. In addition,
more resistant mite populations to coumaphos in Buenos
Aires province have been recently detected (Ruffinengo et
al. 2010). With regard to the results reported in this study,
the presence of amitraz resistance in apiaries where this
acaricide had been used three times in a period of 2 years is
disturbing (Table 2). According to Sammataro et al. (2005),
the presence of resistant mites (in colonies where no strong
acaricides pressures are obvious) may be due to (1) bees
robbing honey from a weak or dying hive (with resistant
mites) within the flight range of the apiary and, as a
consequence, acquiring those mites; (2) introduction of
packaged bees and queens from other states already
parasitized by resistant mites; or (3) drifting bees, a
common phenomenon in large apiaries where phoretic
mites can be swiftly distributed throughout the whole
apiary in a short time.
These results must be a matter of serious concern to all
beekeepers and government authorities. Currently, Argentina
has few mite control alternatives on the horizon. Although,
identification of resistant mites and rotation of chemicals are
needed to limit the spread of the mites (due to: bee transport
across the country for pollination, queens sales, and the
packaged bees, in Argentina) and to prolong the use of the
various acaricides. Based on our findings, it is recommended
that beekeepers began to use some means for assessing the
mite populations before and especially following treatment
with amitraz and coumaphos.
In addition, this study helps in understanding the sources
of resistance to amitraz in the world, due to the few cases
reported until the present day. Other studies on populations
of V. destructor resistant to synthetic acaricides should
continue to carry out with the purpose of obtaining detailed
historical information according to the health status of
different apiaries in all countries. Doing so could generate
stronger conclusions about the obtained results.
In apiaries where Varroa mites are still susceptible,
rotation between amitraz and another miticide should
prolong the effectiveness of these pesticides to prevent the
occurrence of chemically resistant mites. Several studies
reported organic treatments against V. destructor populations
(Eguaras et al. 2003, 2005; Ruffinengo et al. 2005; Damiani
et al. 2010; Maggi et al. 2010b). In apiaries where Varroa
1192
mites are resistant, the introduction of integrated pro-
grams for resistance management is required. This
includes the selection of mite-tolerant bees, monitoring
of mite populations, nonchemical control methods, and
pesticides rotation, whether natural or synthesized.
Acknowledgments The authors would like to thank the UNMDP,
CONICET and CREO (Oportunidades para Conservación, Investiga-
ción y Educación). This research was supported by a grant of
ANPCyT, Picto 443, and PIC 2007 01749 to M.E and by a grant of
CREO to M.M. The experiments comply with the current laws of the
country in which they were performed.
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