Journal of Apicultural Research

ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: http://www.tandfonline.com/loi/tjar20

Multilevel assessment of grooming behavior

against Varroa destructor in Italian and
Africanized honey bees

Ciro Invernizzi, Ignacio Zefferino, Estela Santos, Lucía Sánchez & Yamandú
Mendoza

To cite this article: Ciro Invernizzi, Ignacio Zefferino, Estela Santos, Lucía Sánchez &
Yamandú Mendoza (2016): Multilevel assessment of grooming behavior against Varroa
destructor in Italian and Africanized honey bees, Journal of Apicultural Research, DOI:
10.1080/00218839.2016.1159055

To link to this article: http://dx.doi.org/10.1080/00218839.2016.1159055

Published online: 11 May 2016.

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 Journal of Apicultural Research, 2016
http://dx.doi.org/10.1080/00218839.2016.1159055

ORIGINAL RESEARCH ARTICLE

Multilevel assessment of grooming behavior against Varroa destructor in Italian and
Africanized honey bees
Ciro Invernizzia*, Ignacio Zefferinoa, Estela Santosa, Lucı́a Sáncheza and Yamandú Mendozab
a
Sección Etologı´a, Facultad de Ciencias, Iguá 4225, CP 11400, Montevideo, Uruguay.; bLaboratorio de Apicultura. Instituto Nacional de
Investigación Agropecuaria.(INIA), Ruta 50 km11, CP 70000, Colonia, Uruguay
(Received 9 July 2013; accepted 15 June 2014)

The ectoparasitic mite Varroa destructor is one of the main plagues of honey bees Apis mellifera. Grooming behavior is a
resistance mechanism through which parasitized bees can dislodge mites by themselves (autogrooming) or by the
action of other bees (allogrooming). The objective of this study was to evaluate grooming behavior in Italian (A. m. ligus-
tica) and Africanized (hybrids of A. m. scutellata) bees at the individual, group, and colony levels. Firstly, five behaviors
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were recorded observing bees individually placed on a Petri dish and after placing a mite on their thorax. Secondly, 30
bees of each colony were placed in a Petri dish along with 20 mites and 24 h later fallen mites were counted. Lastly,
the proportion of injured mites collected in the hive floor was determined. At the individual level, Africanized bees
showed a higher total number of reaction behaviors to V. destructor than did Italian bees (U = 182.5; p = 0.02). Groups
of Italian bees could dislodge 60.8 ± 20.0% of mites and Africanized bees dislodged 65.9 ± 15.6% of mites, without
showing significant differences (t = 0.735; p = 0.47). Colonies of Africanized bees showed a higher proportion of injured
mites (29.0 ± 8.6%) than colonies of Italian bees did (17.7 ± 9.8%) (t = 2.92; p = 0.009). Africanized bees are character-
ized by presenting higher resistance to V. destructor than European bees. This study shows that such difference can be,
partly due to grooming behavior. The importance of auto and allogrooming regarding resistance to V. destructor is dis-
cussed.

Evaluación a varios niveles del comportamiento de limpieza contra Varroa destructor en abejas de la
miel africanizadas e italianas

El ectoparásito Varroa destructor es una de las principales plagas de las abejas melı́feras Apis mellifera. El comportamiento
de grooming es un mecanismo de resistencia con el que las abejas parasitadas pueden desprenderse de los ácaros por
sı́ mismas (autogrooming) o por la acción de otras abejas (allogrooming). El objetivo de este estudio fue evaluar el
comportamiento de grooming en abejas italianas (A. m. ligustica) y africanizadas (hı́bridos de A. m. scutellata) a nivel indi-
vidual, grupal y colonial. Primeramente, en abejas ubicadas individualmente en placas de Petri se registró la expresión
de cinco comportamientos de reacción luego de colocarles un ácaro en el tórax. Posteriormente se colocaron en una
caja de Petri 30 abejas de cada colonia con 20 ácaros y 24 horas después se contaron los ácaros caı́dos. Por último, en
cada colonia se determinó la proporción de ácaros mutilados colectados del piso de la colmena. En la evaluación a nivel
individual las abejas africanizadas mostraron un mayor número total de comportamientos de reacción frente a V.
destructor que las italianas (U = 182,5; P = 0,02). Los grupos de abejas italianas consiguieron desprenderse del 60,8 ±
20,0% de los ácaros y los de las abejas africanizadas del 65,9 ± 15,6% de los ácaros, no mostrando diferencias significa-
tivas (t = 0,735; P = 0,47). Las colonias de abejas africanizadas presentaron una mayor proporción de ácaros mutilados
(29,0 ± 8,6%) que las colonias de abejas italianas (17,7 ± 9,8%) (t = 2.92; P = 0,009). Las abejas africanizadas se carac-
terizan por presentar mayor resistencia a V. destructor que las abejas de razas europeas. Este estudio muestra que esta
diferencia puede deberse en parte al comportamiento de grooming. La importancia del auto y el allogrooming en la
resistencia a V. destructor son discutidos.
Keywords: varroosis; resistance; mites; honey bees subspecies; selection

Introduction of new specific chemicals (Elzen, Baxter, Spivak, &

The ectoparasitic mite Varroa destructor colonized the
honey bees (Apis mellifera) and distributed worldwide
during the second half of the twentieth century. Since
then, it has become the main sanitary threat for the
beekeeping industry (Dietemann et al., 2012; Dietemann
et al., 2013; Rosenkranz, Aumeier, & Ziegelmann, 2010).
Controlling V. destructor using synthetic acaricides is
becoming more difficult due to the mite’s acquisition of
resistance against the most used products and the lack
Wilson, 2000; Milani, 1999; Spreafico, Eördegh, Bernar-
dinelli, & Colombo, 2001).
In the search for new tools to control V. destructor,
much research has been done aiming to identify the
resistance mechanisms that honey bees poses (reviewed
in Boecking & Spivak, 1999; Büchler, Berg, & Le Conte,
2010; Rinderer et al., 2010; Rosenkranz et al., 2010).
One of the behavioral resistance mechanisms found is
grooming, by which parasite bees can dislodge mites by

*Corresponding author. Email: ciro@fcien.edu.uy

© 2016 International Bee Research Association

 2 C. Invernizzi et al.
themselves (autogrooming) or receiving help from other
bees (allogrooming) (Boecking & Spivak, 1999). In the
latter, a parasitized bee, by means of a specific dance,
incites its mates to remove mites from its body (Land &
Seeley, 2004). This behavioral resistance mechanism is
readily expressed in the Asian bee Apis cerana, the origi-
nal host of Varroa spp., and contributes to the control
of mite populations (but see Büchler, Drescher, & Tor-
nier1992; Fries, Huazhen, Jin, & Wei, 1996; Peng, Fang,
Xu, & Ge, 1987).
The importance of grooming behavior for controlling
V. destructor is a controversial issue. On the one hand,
grooming behavior is negatively associated to the amount
of mites in the colonies (Andino & Hunt, 2011; Arechava-
leta-Velasco, & Guzman-Novoa, 2001; Boecking & Ritter,
1993; Büchler, 1993; Delfinado-Baker, Rath, & Boecking,
1992; Guzmán-Novoa, Vandame, & Arechavaleta-Velasco,
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2012; Mondragón, Spivak, & Vandame, 2005). On the
other hand, it is doubted whether this behavior has a rele-
vant role in controlling mite population (Harbo & Harris,
1999; Locke & Fries, 2011; Lodesani, Crailsheim, & Mor-
itz, 2002; Rosenkranz & Engels, 1994).
Moretto, Gonçalves, & De Jong, 1993 determined
that the heritability (h2) of grooming behavior is high
(0.71), which would enable improving such resistance
mechanism through artificial selection. However, Stan-
imirovic, Jevrosima, & Nevenka, 2010 assessed grooming
behavior under field conditions along three generations
of queens found moderate to low heritability values
(0.49; 0.18 and 0.16), whereas Ehrhardt, Reinsch, Büch-
ler, Garrido, & Bienefeld, 2007 found even lower values
(<0.15) under similar conditions. Büchler, 2000success-
fully selected colonies showing this resistance behavior
and, after some generations, found more injured mites
as well as lower rates of infection than those found in
colonies under no selection.
The expression of grooming behavior varies among
subspecies of bees. Comparative studies have noticed that
Africanized bees (hybrids of A. m. scutellata) show a more
efficient grooming than European bees (Aumeier, 2001;
Guzman-Novoa et al., 1999; Guzmán-Novoa, Emsen,
Unger, Espinosa-Montaño, & Petukhova, 2012; Moretto
et al., 1993). Such differences could partially explain the
well-known resistance to V. destructor of Africanized bees
in many South and Central American regions (Martin &
Medina, 2004). In the USA, Rinderer et al., 2001 found
that bees brought from the region of Primorsky (Russia),
which presented good resistance to V. destructor, showed
more grooming behavior than bees from Louisiana.
Assessing grooming behavior in bees is problematic.
Both direct observation or video recording of infested
bees in observation hives is difficult, as both methods
are time consuming and do not allow obtaining continu-
ous records (Moretto et al., 1993; Peng et al., 1987;
Thakur, Bienefeld, & Keller, 1997; Vandame, Morand,
Colin, & Belzunces, 2002). Thus, evaluating grooming
behavior in bees of a given colony is usually done indi-
rectly by considering the proportion of injured mites
(with mutilated legs and occasionally cuts in the idio-
soma) collected in the hive floor (Bienefeld, Zautke,
Pronin, & Mazeed, 1999; Guzmán-Novoa et al., 2012;
Mondragón et al., 2005; Rosenkranz, Fries, Boecking, &
Sturner, 1997 ;Ruttner & Hänel, 1992; Vandame et al.,
2002). Nevertheless, a proportion of injured mites can
be product of the cleaning of cells with infested brood
(hygienic behavior) (Rosenkranz et al., 1997) or caused
by predators (Bienefeld et al., 1999), or by the moth
Galeria mellonella (Szabo & Walker, 1995). Marks on the
idiosoma of mites can be the result of malformation
(Davis, 2009), so they should not be necessarily inter-
preted as a consequence of bee grooming behavior.
The expression of grooming behavior is also affected
by environmental factors. Currie & Tahmasbi, 2008
found that bees selected for high and low expression of
grooming behavior kept their differences at 25 and
34 ˚C, but not at 10 ˚C. Büchler, 1993 and Moosbeck-
hofer, 1997 found that the proportion of injured mites
presented seasonal variation.
Grooming behavior has been assessed under con-
trolled conditions in small groups of bees infested by V.
destructor confined in boxes (Andino & Hunt, 2011; Are-
chavaleta-Velasco & Guzman-Novoa, 2001; Currie &
Tahmasbi, 2008) or observing the behavior of a bee
placed in a Petri dish after placing a mite on its body
(Aumeier, 2001; Guzmán-Novoa et al., 2012).
In Uruguay, most bees are hybrids, crosses between
European A. m. mellifera and African A. m. scutellata bees,
and almost 100% of African haplotypes can be found in
the borderline with Brazil following a southward
decreasing gradient (Burgett, Shorney, Cordara, Gardiol,
& Sheppard, 1995; Collet, Ferreira, Arias, Soares, & Del
Lama, 2006; Diniz, Soares, Sheppard, & Del Lama,
2003). Recently, Branchiccela et al., 2014 showed that
Africanized bees are completely established in Uruguay,
representing 80% of the analyzed colonies, distributed
throughout the whole country. However, in the last
years, beekeepers have introduced a great amount of
queens of Italian bees A. m. ligustica, mainly in the west-
ern littoral provinces, taking advantage of the gentleness
of this bee subspecies (Branchiccella et al., 2014).
Most Uruguayan beekeepers control varroa using
conventional synthetic acaricides in autumn (Invernizzi
et al., 2011). Nevertheless, there are still some areas in
the East and North of the country where bees are
much Africanized and there is little beekeeping activity,
and where V. destructor does not cause severe damage
(a loss of untreated colonies less than 30%; Yamandú
Mendoza, unpublished data).
The aim of this study was to evaluate grooming
behavior in Africanized and Italian bees at the individual,
group, and colony levels.
Materials and methods
Studies were conducted in February and March in 2011
(summer in the southern hemisphere) at the experimen-

tal station “La Estanzuela” of the Instituto Nacional de
Investigación Agropecuaria (Colonia Province, Uruguay).
Grooming behavior was evaluated in Africanized and
Italian bees at the individual, group, and colony levels.
Africanized bees were collected at Treinta y Tres Pro-
vince (east region of the country), where there is an
important Africanization of bees and good tolerance to
V. destructor. Italian bees were provided by a beekeeper
from Colonia Province who imported queens of this
subspecies from the Buenos Aires Province (Argentina).
Adult female mites were obtained from the brood of
heavily infested colonies, and kept in Eppendorf tubes,
3–5 h before starting the evaluations.
Experiment 1. Evaluation of grooming behavior at
the individual level
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In order to observe the behavioral response of a bee
when a mite contacts it, 24 Italian and 24 Africanized
bees were put each in a separate Petri dish (10-cm
diameter). They were obtained from six colonies of
each subspecies (four bees of each colony, collected
from brood combs). A mite was placed on the dorsal
part of the thorax of a bee using a small brush, and then
the bee was observed for two minutes. We recorded
the occurrence of five reactions which compose the
grooming behavior, similar to those described by:
Aumeier, 2001
Cleaning: cleaning antennae and body with legs.
Shaking: shaking the abdomen
Biting: trying to remove the mite using the mandibles
Rolling: doing a somersault while hunting the mite
and using all pairs of legs.
Attempting to fly
Our study did not consider the classification done
by Aumeier, 2001 in which Cleaning and Biting was weak
or intense depending on whether they lasted less or
more than 5 s, respectively.
We recorded whether reactions occurred in the
first or second minute, and if the mite was removed in
the observation time.
Experiment 2. Evaluation of grooming behavior at
the group level
Grooming at the group level was evaluated in 13 colo-
nies of Italian bees and 13 of Africanized bees. Thirty
bees were taken from brood combs and placed in a
Petri dish, covered by a 3-mm mesh screen. Bees were
exposed to low temperatures (10–15 min at 4 ˚C) in
order to manipulate them easier. A 1-cm diameter ball
of candy (honey and powdered sugar) was put in each
Petri dish to feed the bees. Once all the bees were in
the dish, 20 mites were added through the screen using
a small brush. When all mites were on the bees, the
dish was inverted and put on the lid which served as
the floor of the device. The lid was covered with a
paper spread with petroleum jelly to pick up the fallen
Grooming behavior against Varroa destructor 3
mites. Bees were incubated for 24 h at 28 ˚C and then
the number of dead bees was recorded. Fallen mites
were observed using a binocular microscope (40x) to
record whether they had mutilated legs. When more
than 10% of bees were dead, evaluations were not
taken into account and the experiment was repeated.
Experiment 3. Evaluation of grooming behavior at
the colony level
Grooming at the colony level was evaluated in 10 colonies
of Italian bees and 10 colonies of Africanized bees. The
bees filled completely the brood chamber of Langstroth
hives. A 3-mm mesh screen was used as the hive floor, and
this had a tray under it that had a paper coated with petro-
leum jelly to retain fallen mites (Guzmán-Novoa et al.,
2012). Fallen mites were collected for 10 days, and 30
mites were observed using a binocular microscope (40x)
to determine the proportion of mites with mutilated legs.
Statistical analyses
All data were analyzed to check for normal distribution
and homogeneity of variances. If both conditions were
met, then parametric tests were performed. In Experi-
ment 1, grooming behavior was quantified as the num-
ber of Italian and Africanized bees which reacted to V.
destructor. A Chi-square test was used to analyze
whether there were differences between subspecies,
and between the number of bees which reacted to the
mite in the first and second minute. Grooming was also
measured as the average number of times a bee mani-
fested each behavioral reaction toward the mite. Mann–
Whitney test was used to check for differences between
subspecies and also between the number of bees which
reacted to the mite in the first and second minute.
In Experiment 2, grooming behavior in colonies was
quantified as the number of fallen mites over the total
number of mites. A Student’s t-test was used to check
for differences of mean values between groups of Italian
and Africanized bees. In Experiment 3, grooming behav-
ior of the colonies was quantified as the percentage of
injured mites. Differences between subspecies were ana-
lyzed using the Student test for mean values. Finally, a
Spearman linear correlation was done between the per-
centage of dislodged mites in the laboratory test (Exper-
iment 2) and the percentage of mutilated mites in the
field test (Experiment 3) using only the results obtained
in colonies included in both experiments (8 Italian and
10 Africanized). All statistical analyses were considered
as significant when P was < 0.05.
Results
Experiment 1. Evaluation of grooming behavior at
the individual level
Cleaning, Shaking, Biting, Rolling, and Attempting to fly
were observed with low frequency in the 48 Italian and
 4 C. Invernizzi et al.
Africanized bees after placing a mite on their thorax.
Notably, four Africanized bees were observed beating
their wings throughout the two minutes of observation.
Sixteen Italian and 19 Africanized bees presented at
least one of the reactions to V. destructor; these frequen-
cies did not differ between subspecies (Chi
square = 0.95; df = 1; p = 0.33). The number of bees
that reacted to the mite in the first and second minute
of the observation time was 10 and 9, respectively, for
Italian bees, and 15 and 12 for Africanized bees. Within
each subspecies, no significant difference was found in
the number of bees that reacted in each minute of
observation (Chi square = 0.09; df = 1; p = 0.77, for
Italian bees; Chi square = 0.76; p = 0.38, for Africanized
bees).
There were no significant differences between sub-
species of bees in the number of times each of the five
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reactions was observed (Cleaning: U = 239; p = 0.26;
Shaking: U = 243; p = 0.26; Biting: U = 288; p = 0.98;
Rolling: U = 251; p = 0.22; Attempting to fly: U = 286;
p = 0.96; Figure 1). However, Africanized bees showed
an overall higher number of reactions to V. destructor
than Italian bees (U = 182.5; p = 0.02) (Figure 1).
Overall, bees that manifested any response to V.
destructor did it between one and six times in an obser-
vation period, and repeated it up to four times. In bees
of both subspecies, Cleaning and Shaking were the most
frequent responses, i.e., 60% of the total reactions
observed (Figure 1). Only two Italian and two African-
ized bees (8% out of the total) bit mites, and only two
Italian and one Africanized bees (6.3% out of the total)
dislodged the mite successfully.
There were no significant differences in the average
number of reactions to V. destructor between the first
and second minute of observation for Italian and
Africanized bees (U = 271.5; p = 0.70 and U = 249;
p = 0.40, respectively).
Italians bees
4 P = 0,025
Reactions per bee
2 n.s.
n.s.
1 n.s.
n.s.
0
Cleaning Shaking Biting
Figure 1. Frequency of the reactions Cleaning, Shaking, Biting,
Rolling, and Attempting to fly in Italian and Africanized bees, in
two minutes of observation after placing a mite on their tho-
rax.
Notes: ns: not significant differences for Mann–Whitney test
(p > 0.05).
Experiment 2. Evaluation of grooming behavior at
the group level
Groups of Italian bees dislodged 60.8 ± 20.0% of the
mites while groups of Africanized bees did 65.9
± 15.60%, but this difference is not significant (t = 0.735;
p = 0.47). No mutilated legs were observed in any of
the 312 fallen mites.
Experiment 3. Evaluation of grooming behavior at
the colony level
The analysis of fallen mites showed that Africanized bee
colonies contained a higher proportion of mites with
mutilated legs (29.0 ± 8.6%) than Italian bee colonies
(17.7 ± 9.8%) (t = 2.92; p = 0.009).
Finally, there was no association between the num-
ber of mites dislodged in the laboratory tests (Experi-
ment 2) and percentage of injured mites collected in the
artificial hive floor (Experiment 3), taking into account
the 18 colonies used for both experiments (8 of Italian
bees and 10 of Africanized bees) (r = 0.24; p = 0.34).
However, a significant correlation was found (r = 0.48;
p = 0.05) when one colony was removed from the anal-
ysis (Figure 2). Such colony showed a response outlying
the tendency of the remaining colonies of the group,
i.e., it had the largest percentage of the mites dislodged
in Experiment 2 and the least injured mites in Experi-
ment 3.
Discussion
Africanized bees have been reported to have higher
resistance to V. destructor than European (Camazine,
1986; De Jong, 1997; Moretto, Gonçalves, De Jong, &
Bichuette, 1991; Rosenkranz et al., 2010). Grooming
could be one trait which contributes to resistance of
Africanized bees. In this study, grooming was assessed in
Italian bees (A. m. ligustica) and Africanized bees (cross-
ings of European bees, mainly A. m. mellifera, with Afri-
can bees A. m. scutellata) at the individual, group, and
Africanized bees colony levels.
n.s.
Rolling Attempting Sum of
to fly reactions
Figure 2. Spearman linear correlation between the percent-
age of dislodged mites in the laboratory test (Experiment 2)
and the percentage of mutilated mites in the field test
(Experiment 3) (r = 0.48; n = 17; p = 0.05).

In the first experiment, the five reaction behaviors
reported by, Aumeier, 2001 Cleaning, Shaking, Biting,
Rolling, and Attempting to fly, were recorded while
observing solitary bees infested by females V. destructor.
The low frequency with which these behaviors occurred
coincides with that reported by Aumeier, 2001 and can
be what prevented detecting differences between Italian
and Africanized bees. Nevertheless, when all behaviors
were grouped, we found that Africanized bees reacted
more to V. destructor than Italian bees did. Aumeier,
2001 analyzed the data similarly and found that African-
ized bees reacted more frequently than European bees
(A. m. carnica) when V. destructor females were placed on
their thorax. Guzmán-Novoa et al., 2012 used an analo-
gous methodology to the one used in our study to com-
pare the intensity of grooming in bees with genotypes
resistant and susceptible to V. destructor. They found
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that the behavior was expressed with higher intensity in
the former ones, and that such intense grooming was
more efficient to dislodge mites than a mild one. In the
same study it was verified that Africanized bees have a
higher ability to dislodge a mite than Italian bees do.
In this study, four Africanized bees beat their wings
constantly during the observation period, a behavior not
reported by Aumeier, 2001. Beating wings is one of the
responses of bees toward ants (Fell, 1997). It is possible
that bees infested by V. destructor beat their wings as a
similar reaction to that recorded toward ants.
Out of the 48 bees observed, only 3 (6.3%) (2 Italian
and 1 Africanized) could dislodge the mite, which is less
than found by Aumeier, 2001 and Guzmán-Novoa et al.,
2012 using the same technique (though with an addi-
tional minute of observation). Aumeier, 2001 found that
18.3% of Africanized bees and 9.1% of Carniolan bees
could remove the mite. Guzmán-Novoa et al., 2012
found that between 15.0 and 34.9% of the bees bearing
resistant genotypes could dislodge the mite (29.4% in
Africanized bees), while 4.3 (Italian bees) to 16.2% of
bees bearing susceptible genotypes could do it. Marked
differences in the proportion of bees that could dislodge
the mite between our study and those of Aumeier,
2001 and Guzmán-Novoa et al., 2012 could be due to
little methodological differences which greatly affect the
ability either of bees to dislodge the mite or of the mite
to avoid bees’ attempts of being removed.
In the second experiment, groups of Italian and
Africanized bees artificially infested with mites were put
in Petri dishes. It was found that the number of
removed mites was somewhat superior to 60%, without
detecting differences between subspecies groups. This
result contrasts the one obtained in the first experi-
ment, in which Africanized bees showed more reaction
behaviors to V. destructor than Italian bees.
The analysis of fallen mites did not show any indi-
viduals with mutilations, as expected for mites col-
lected from hive floors (typically between 10 and 30%)
(Bienefeld et al., 1999; Guzmán-Novoa et al., 2012;
Mondragón et al., 2005; Rosenkranz et al., 1997; Rut-
Grooming behavior against Varroa destructor 5
tner & Hänel, 1992; Experiment 3 of this study). In
colonies under natural conditions, it is likely that most
mites dislodged by bees are not injured and fall on
other bees without any consequences regarding mite
removal of the colony. If mites fall on the hive floor,
these can probably walk up and get in touch with bees
again. In this sense, one of the authors of this study
(Estela Santos) has observed that, under laboratory
conditions, females V. destructor can walk vertically
enough so as to climb the hive floor up to the nest
without any difficulty, and get in contact with the bees
again. Nonetheless, those mites with mutilated appen-
dixes will probably have difficulties in grabbing to the
bee body, have more exposure to being dislodged and
end up dying as a result of injuries. Thus, when fallen
mites are collected from artificial hive floors, the
observed proportion of injured/not injured mites is
likely to be higher than that expected for all mites
removed by bees.
Evaluation of grooming using little groups of bees
artificially infested with V. destructor allows discarding
environmental factors not controlled under field condi-
tions (nectar flow, infestation level, environmental tem-
perature, colony size, etc.). Using this technique, Currie
& Tahmasbi, 2008 got, through a bidirectional selection,
bees that express high and low efficiency for grooming
behavior.
In the last experiment, grooming behavior in colo-
nies of Italian and Africanized bees was evaluated using
a conventional methodology, i.e., observing damage in
fallen mites. Africanized bee colonies presented almost
twice as many injured mites as Italian bee colonies.
These results match those reported by Moretto et al.,
1993; Aumeier, 2001; Vandame et al., 2002; and Guz-
mán-Novoa et al., 2012 and those found in Experiment
1 of this study, in which grooming of Africanized and
European bees was assessed using a variety of method-
ologies.
Differential resistance to varroosis by Africanized
and European bees is often explained through factors
such as hygienic behavior, grooming behavior, brood
period, duration of the brood cycle, cell size, swarming
frequency, brood attractiveness (De Jong, 1997; Rosenk-
ranz et al., 2010). Results obtained herein, along with
those of Moretto et al., 1993; Aumeier, 2001; Vandame
et al., 2002 and Guzmán-Novoa et al., 2012 show that
grooming can be a factor of main importance to explain
higher resistance of Africanized bees to V. destructor.
Our results show that bees in Uruguay, which have
a significant degree of Africanization, express grooming
behavior against V. destructor in a more efficient way
than Italian bees do. Since varroosis is the main sanitary
problem in honey bees of the country (Invernizzi et al.,
2011) caution should be taken when entering and repro-
ducing Italian bees. On doing so, there is risk of weak-
ening the behavioral resistance in local populations of
Africanized bees due to the impossibility of preventing
crossings among different bee subspecies.
 6 C. Invernizzi et al.
Grooming is a behavioral resistance mechanism
honey bees present against V. destructor mites, likely to
be selected in order to gain tolerance against the para-
site (Büchler, 2000). When implementing a methodology
to evaluate a valuable heritable characteristic, the influ-
ence of environmental factors should be taken into
account as well as its practicality and economy. Either
observing the behavior of isolated bees after getting in
touch with V. destructor under laboratory conditions, or
quantifying the number of mites a confined group of
artificially infested bees can dislodge, stand out as
promising techniques to indirectly assess grooming
behavior of colonies. Still, these evaluations are time
consuming and require obtaining female V. destructor
mites to infect bees, so they would be restricted to
research groups and professional technicians who have
the necessary resources. However, Andino & Hunt,
Downloaded by [Orta Dogu Teknik Universitesi] at 09:47 12 May 2016
2011 have recently put forth a more accessible tech-
nique to assess grooming behavior at the laboratory.
This technique consists in introducing a frame with bees
from the colony in a cage (52.5 27 7.2 cm) with a
metal screen bottom. After 72 h, the fallen mites and
the mites remaining on the bees are counted. The pro-
portion of dropped mites is positively correlated with
the number of mutilated mites in the colony.
A problem that has not been approached yet is the
estimation of the relative importance auto and
allogrooming have in dislodging V. destructor from the
body of the bees. Through different behavioral compo-
nents of autogrooming (in which the main are cleaning
using the legs and shaking), bees may be able to remove
the mite from its body without damaging it. Instead, if a
bee used its mandibles to dislodge the mite from
another bee, it could end up in lethal mutilations for the
mite. Hence, a standard evaluation of grooming behavior
of a colony, in which the proportion of injured mites is
quantified, may only indicate the magnitude of
allogrooming in the colony. Future studies should be
addressed to elucidate the weight of individual (auto-
grooming) and social (allogrooming) responses, as well
as the relationship between both of them, in the behav-
ioral resistance against V. destructor.
Acknowledgments
We thank Matı́as Maggi for helpful comments on the manu-
script.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by the Instituto Nacional de
Investigación Agropecuaria.
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