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Apidologie Available online at:
c
INRA/DIB-AGIB/EDP Sciences, 2010 www.apidologie.org
DOI: 10.1051/apido/2010011 Review article
Breeding for resistance to Varroa destructor in Europe*
Ralph B¨
uchler1,StefanBerg2,YvesLeConte3
1Landesbetrieb Landwirtschaft Hessen, Bieneninstitut, Erlenstr.9, 35274 Kirchhain, Germany
2Bayerische Landesanstalt für Weinbau und Gartenbau, Fachzentrum Bienen, An der Steige 15,
97209 Veitshöchheim, Germany
3INRA, UMR 406, Abeilles et Environnement, Laboratoire Biologie et Protection de l’Abeille, Site Agroparc,
Domaine Saint-Paul, 84914 Avignon, France
Received 27 October 2009 – Revised 29 January 2010 – Accepted 30 January 2010
Abstract – The rich variety of native honeybee subspecies and ecotypes in Europe offers a good genetic
resource for selection towards Varr oa resistance. There are some examples of mite resistance that have de-
veloped as a consequence of natural selection in wild and managed European populations. However, most
colonies are influenced by selective breeding and are intensively managed, including the regular use of miti-
cides. We describe all characters used in European breeding programs to test for Va r ro a resistance. Some of
them (e.g., mite population growth, hygienic behavior) have been implemented in large-scale selection pro-
grams and significant selection effects have been achieved. Survival tests of pre-selected breeder colonies
and drone selection under infestation pressure are new attempts to strengthen effects of natural selection
within selective breeding programs. Some perspectives for future breeding activities are discussed.
Varroa resistance /breeding program /tolerance character /vitality /natural selection
1. INTRODUCTION
Europe has a high diversity of climatic re-
gions and natural biotopes. As honeybees are
endemic to most of regions in Europe, they
have evolved into numerous natural subspecies
and ecotypes (Ruttner, 1988) with different
adaptive capacities. This diversity offers rich
potential genetic resources for selection on
mite resistance. Wild honeybee populations
under natural selection are very rare, not only
as a consequence of Va r roa infestation but also
due to a lack of natural habitats in densely pop-
ulated areas with intensive use of farmland and
forests. Today,nearly the entire honeybeepop-
ulation of Europe is managed by the beekeep-
ing industry, which involves regular chemical
treatments for diseases and mites that can con-
Corresponding author: R. Büchler,
ralph.buechler@llh.hessen.de
* Manuscript editor: Marla Spivak
tribute to the propagation of susceptible bee
populations.
Breeding and selection techniques have a
long tradition in European countries and are
widely used with varying degrees of rigor.
Starting at the end of the 19th century,
beekeepers transported queens and colonies
across the natural ranges of different sub-
species and ecotypes. This practice led to hy-
bridisation followed by changes in the spread
and frequency of certain genotypes. While the
economically important subspecies A. m. car-
nica and A. m. ligustica are now widely spread
throughout Europe, the native populations of
A. m. mellifera,A. m. siciliana,A. m. mace-
donica, and other European subspecies have
been diminished and partly extinguished from
their natural ranges (Meixner et al., 2010).
Breeding programs are well established in
many European countries, based on perfor-
mance tests, statistical data analysis, and the
mass propagation and controlled mating of
Article published by EDP Sciences
2 R. Büchler et al.
selected breeder colonies (Lodesani and Costa,
2003). The strong impact of selective breed-
ing on the population is evident due to the
significant reduction of genetic variability in
European compared to wild African colonies
(Moritz et al., 2007).
In the majority of selection and breeding
programs, economic traits (such as honey pro-
ductivity and colony strength) together with
traits desirable for modern beekeeping (such
as gentle temper and low swarming tendency)
have been of predominant importance. In con-
trast, disease resistance, viability, and adapta-
tion to local conditions were considered less
important, as deficiencies in these characters
could often be compensated by pharmaceuti-
cals, artificial feeding, and other management
techniques.
Since Varroa destructor began spreading
throughout Europe, the beekeeping industry
has had to face a new situation. The regu-
lar use of chemical treatments has been ac-
companied by several disadvantages, such as
high costs and labor, residues in bee products,
and the selection of mites resistant to acari-
cides. However, repeated high colony losses
due to varroosis could not be prevented. Con-
sequently, research on mite resistance of honey
bees started in the 1980s and continues to re-
ceive a large amount of scientific interest and
practical attention in Europe.
We consider resistance in honey bees as the
ability of a bee population to survive without
therapeutic treatments in a given environment
and management system. Resistance is there-
fore not an absolute trait, but rather has to be
viewed as the result of successful interactions
in a specific environment.High levels of resis-
tance occur in some untreated European bee
populations. However, as most colonies are
under strong influence of modern beekeeping
management, which includes regular use of
acaricides and requeening with selected stock,
Varro a resistance should be implemented on a
broader scale in selective breeding activities.
This implementation depends on having suit-
able test characters and an effective coordina-
tion of breeding programs. Comparative tests
between European bee strains and Primorski
lines from the US allow for an evaluation of
the European breeding efforts.
2. MITE RESISTANCE
IN UNTREATED EUROPEAN
BEE POPULATIONS
Varro a surviving bee (VSB) colonies were
identified in untreated Apis mellifera popula-
tions from a few different locations in Europe.
Similar observations were reported by Seeley
(2007) from a small bee population in the
Arnot Forest in the northeastern United States.
When Va r ro a mites first invaded France in
the 1980s, feral and untreated colonies were
destroyed by the mites. The first observations
of natural VSB were made in 1994 in west-
ern France, near Le Mans, where feral and un-
treated colonies seemed to survive the mite in-
festation for a few years. In 1999, 10 out of 12
of such untreated colonies were still surviving
(5 of them survived for more than 11 years).
At that time, 82 colonies that were untreated
for at least 2 years were collected to character-
ize their survival without Varro a control. The
colonies were managed only to monitor their
survival. They were allowed to swarm and re-
place their queens naturally. On average, the
survival of those colonies was 7.88 ±0.3 years
with a maximum of 15 years (Le Conte et al.,
2007).
Varro a populations were estimated between
VSB and Va r ro a susceptible (control) colonies
by counting natural mite mortality using a
screened bottom board to collect the mites (see
Fries et al., 1991). Traps were left in place con-
stantly, and the mites falling down on the bot-
tom board were counted one to three times a
week depending on the amount of brood in the
colonies. In Avignon, from April 2002 to June
2004, natural mite mortality was observed in
12 VSB colonies and 16 control (Var roa sus-
ceptible) colonies.
The number of mites collected in VSB
colonies was three times lower than in con-
trol colonies, which was statistically signifi-
cant (Le Conte et al., 2007), and each year
the highest differences were observed between
March and June (Fig. 1). The infestation of
the VSB colonies from July to September,
as measured by mite fall, decreased in both
groups. After that period, Va r ro a populations
increased more rapidly in control colonies than
in VSB colonies. The VSB maintained lower
Breeding for resistance to Varroa destructor in Europe 3
Figure 1. Number of mites collected per month on the bottom board (mean ±S.E.) from April 2002 to June
2004. Mites were not counted between October 2003 and December 2003. ♦represent mean number of
mites in VSB (n =12 colonies), represent control colonies (n =16). (* Significant statistical differences,
Mann-Whitney test, P<0.05).
Varro a populations year round, suggesting that
VSB colonies had developed mechanisms to
inhibit the growth of Varro a populations.
Various hypotheses could explain this phe-
nomenon. First, the bees may have developed
behavioral or physiological resistance against
the mite. Martin et al. (2002) showed that the
VSB have better ability to recognize the mite
compared to control bees. Thus, VSB could be
more efficient in their ability to detect and de-
stroy the mite from workers through groom-
ing behavior, similar to the behavior of Apis
cerana (Peng et al., 1987). They may also
be more efficient in removing mite-infested
pupae from the cells as reported in the MN
Hygienic stock (e.g., Boecking and Spivak,
1999; Spivak and Reuter, 1998a, 2001)and
Varro a sensitive hygienic (VSH) bee strains
from Louisiana (Harbo and Harris, 2005a). In-
deed, preliminary observations show that VSB
are able to detect and remove mite-infested pu-
pae from cells (Anderson and Le Conte, pers.
obs.). Interestingly, gene-expression analysis
of the VSB shows over-expression of a set
of genes related to responsiveness to olfac-
tory stimuli compared with Va r ro a suscepti-
ble bee colonies (Navajas et al., 2008). Harbo
and Harris (2005b) suggested only a few
genes to be involved in VSH behavior, which
seems to be widespread in honeybee pop-
ulations. The quantitative trait loci (QTLs)
for hygienic behavior from the MN Hygienic
stock (Lapidge et al., 2002) were recently
confirmed, and some genes regulating this be-
havior are thought to be involved in olfactory
sensitivity (Oxley et al., in press). The genes
involved in olfaction in the VSB, VSH, and
MN Hygienic stocks are potentially very inter-
esting to characterize as they may be involved
in the ability of these bees to detect and re-
move and thus resist Varro a .
Other characters – such as the inhibition
of Varro a reproduction, the duration of the
post-capping stage, and thermoregulatory abil-
ities – may also contribute to the survival of
the VSB. Moreover, an average of 41.5% of
the VSB colonies swarmed each year. Fries
et al. (2003) demonstrated that swarming af-
fects Varro a populations in a colony for at least
one year; thus, swarming could at least partly
explain the survival of the VSB.
Differential virulence of the mite also can
be hypothesized to explain the survival of
VSB. After the first years of Va r roa invasion
in France, most of the untreated colonies were
found dead with plenty of mites trapped in en-
tire frames of dead sealed brood. Individual
fitness of a mite in those cells was therefore
4 R. Büchler et al.
null. As a less-virulent parasite (such that it
would not kill the host and would have an
increased individual fitness), the hypothesis of
sub-populations of mites with different levels
of virulence was tested using mitochondrial
and nuclear microsatellites markers (Navajas
et al., 2002; Solignac et al., 2003). The struc-
ture of Va r roa population in Europe was found
to be an invasive clone (Solignac et al., 2005).
Therefore, it is unlikely that sub-populations
of less-virulent mites could explain VSB, or
if they are, virulence would be due to a lim-
ited number of genes as it is the case with Var-
roa populations that are resistant to the acari-
cide fluvalinate-tau (Milani, 1999; Liu et al.,
2006).
Acute paralysis virus (APV) and deformed
wing virus (DWV) are resident in bee colonies
and become more pathogenic when associ-
ated with Va r ro a (Sumpter and Martin, 2004).
Therefore, survival of VSB could be due to a
higher tolerance of the bees to those viruses.
This hypothesis was tested, and data have
shown that the VSB had less APV and CPV
(chronic paralysis virus) compared to control
bees. However, the VSB did not survive longer
compared to control bees when injected with
the two viruses (Le Conte, personal communi-
cation). This suggests that the VSB have fewer
viruses because they have fewer mites to trans-
mit virus in the bee population. Nevertheless,
it is reasonable to suggest that honeybee resis-
tance, Varro a virulence, and virus prevalence
are constantly under selective pressure, and
that natural selection favors a co-evolution that
secures the survival of both the host and para-
sites.
The presence of a specific pathogen of the
Varro a mite, such as fungus, cannot be ruled
out and should be investigated as a factor con-
tributing to the survival of VSB. Also, the
effect of the environment and apicultural meth-
ods on the survival of VSB cannot be ex-
cluded. The areas where the experiments were
done are outside France’s major agricultural
zone and are very favorable to the develop-
ment of honeybee colonies. The colonies were
manipulated only if necessary and were not
moved or managed as professional beekeeping
would recommend.
3. BREEDING FROM UNTREATED
SURVIVOR COLONIES
The occurrence of VSB in natural Euro-
pean populations led to the study of survival in
other infested honeybee colonies that were not
treated for Va rro a mites in order to select for
Varro a resistance. This approach, called the
Bond test (“Live and let die!”), has been used
successfully in France (Kefuss et al., 2004)
andSweden(Friesetal.,2006).
Kefuss et al. (2004) initiated the first Bond
test in 1993 on 12 Apis mellifera intermissa
colonies imported from Tunisia in France,
near Toulouse. The resistance of those bees
was compared with 12 A. m. carnica Varroa-
susceptible colonies after exposure to heavy
Varro a infestations. Only A. m. intermissa
colonies survived. They hybridized with lo-
cal bee populations and most of the hybrids
survived mite infestations indicating a genetic
control of the resistance.
Since 1999, Kefuss et al. (2009) have not
used any treatments against Va rro a in their
professional beekeeping enterprise. From this
naturally surviving stock, they subsequently
select their breeder colonies for economical
traits. The best colonies are then tested for hy-
gienic behavior (using a freeze-killed brood
assay) and for Varro a infestation. Their colony
losses are comparable to other beekeepers of
the region that still treat their hives with aca-
ricides. Adult bee infestation usually remains
below 5% and, according to their report, does
not economically justify the use of chemicals.
In Gotland, an island of the Baltic Sea, Fries
et al. (2006) described the survival of 150
honey bee colonies that were subject to the
Bond test. Five of the colonies survived over 5
years. Later, Fries and Bommarco (2007)com-
pared the surviving colonies to Varro a suscep-
tible bees parasitized by mites from a different
source. The mite load in the Bond colonies was
82% lower compared to the control colonies. It
was hypothesized that the differences in mite
populations may have been due to the smaller
brood areas in the Bond colonies and differ-
ences in mite distribution, with more mites
present on adults rather than the brood in the
Bond colonies.
Breeding for resistance to Varroa destructor in Europe 5
Fries et al. (2006) pointed out that when
beekeepers treat colonies to control the mite,
they remove the selective pressure on both the
host and the parasite, which could result in
co-evolution with long term survival of both.
The presence of wild colonies/swarms and un-
treated apiaries can promote co-evolution that
could spread to larger areas. Even if those sur-
viving colonies may not be interesting for pro-
fessional beekeeping because of their lower
honey production due to the cost of the toler-
ance (Le Conte et al., 2007), they can form the
basis for integrated Varro a management and
selection programs to increase honey produc-
tion.
4. RESISTANCE CHARACTERS
FOR THE USE IN SELECTIVE
BREEDING PROGRAMS
Because the effects of natural selection
on increasing resistance to the mites have
been widely eliminated by modern beekeep-
ing practices (through the regular use of aca-
ricides and requeening with productive and
gentle breeding lines that have low swarming
tendency), the development of genetic resis-
tance depends on successful implementation
of breeding programs that use suitable resis-
tance characters in the selection process of
honeybees.
The selection for mite resistance in treated
populations has to rely on indirect selection
characters, because the direct trait of surviv-
ability cannot be studied when the colonies
are influenced by medical treatments. Start-
ing about 25 years ago, much research in
European institutes focused on the identifica-
tion of suitable selection characters (Büchler,
1994b). In addition to the biological relevance
(correlation with the direct selection goal), the
heritability and the practicability of testing un-
der field conditions were deemed to be of ma-
jor importance in the implementation of such
characters in breeding programs.
In the following, we briefly summarize the
research on characters that have been used –
at least temporarily – in European breeding
projects for increased mite resistance.
4.1. Mite population development
The effect of varoosis on the colony level
depends on the infestation level (Garrido and
Büchler, unpubl. data; Genersch et al., 2010).
Therefore, slow and limited mite population
growth is a fundamental criterion of resistant
stock that can be used as a character for selec-
tive breeding on mite resistance, even if the un-
derlying behavioral and physiological causes
remain unknown. Different methods to accu-
rately estimate the Va r ro a infestation level are
well documented (Fuchs, 1985; Rademacher,
1985; Calatayud and Verdu, 1993; Garza and
Wilson, 1994;Brancoetal.,2006). How-
ever, methods used for the comparison of
test colonies in large-scale selection programs
need to be simple, reliable, and well standard-
ized to optimize the heritability of the infesta-
tion data. Based on the number of mites killed
by an annual Apistantreatment (Pechhacker,
1992), Boigenzahn and Willam (1999) esti-
mated the heritability of infestation to be h2=
0.13. The heritability can be increased if the
test is started with a uniform artificial infesta-
tion of all colonies (Büchler, 1994a, 2000)but
requires too much effort for routine field selec-
tion.
In the AGT breeding program (“Arbeits-
gemeinschaft Toleranzzucht”, see below) mite
population growth during the brood season is
used instead of measures of absolute infes-
tation level. For each colony, the infestation
of a bee sample (at least 30 g per hive) col-
lected in the first 10 days of July is com-
pared to the natural mite fall during 3–4 weeks
in spring (during Salix bloom as a phenolog-
ical standardization for different climatic re-
gions). The bee sample is taken from honey
combs of the uppermost box, since they have a
more uniform infestation (repeatability of in-
festation was 0.85 from honey combs versus
0.63 from samples taken from the hive en-
trance and 0.74, from the central brood nest;
Garrido and Büchler, unpubl. data). Prelim-
inary estimates on the basis of about 5000
colonies show median heritability values for
this method of monitoring the mite population
growth (Ehrhardt and Bienefeld, unpubl. data).
The technique is easy and flexible in practice
(independent of colony treatment before and
6 R. Büchler et al.
after the test period) and, in addition to its use
in the AGT breeding program, is used rou-
tinely in several European countries.
4.2. Hygienic behavior
Hygienic behavior towards infested worker
brood cells has been recognized as a vital com-
ponent of Va r ro a resistance in its original host
A. cerana (reviewed by Rath, 1999)andas
an antiseptic behavior against various brood
diseases, including varroosis, in A. mellifera
(reviewed by Boecking and Spivak, 1999;
Wilson-Rich et al., 2009). The performance of
colonies regarding this trait has been success-
fully improved by selective breeding, and it
has been shown to be an effective measure to
improve resistance of A. mellifera against Va r-
roa, AFB, EFB, and chalkbrood (Spivak and
Reuter, 1998a; Palacio et al., 2000; Harbo and
Harris, 2005a; Ibrahim et al., 2007; Büchler
et al., 2008; see also Rinderer et al., 2010,for
further details and references).
Different methods have been developed
to test brood-hygiene behavior under stan-
dardized conditions, including the assay-
ing the bees response to Va r roa infested
brood cells (Boecking and Drescher, 1992;
Hoffmann, 1996) as well as freeze-killed
(Momot and Rothenbuhler, 1971; Spivak and
Reuter, 1998b) and pin-killed brood (Newton
and Ostasiewski, 1986;Hoffmann, 1996).
These methods differ in the effort required and
results, and their suitability for selective breed-
ing programs is being discussed. While the
freeze-killed brood assay is recommended as
a more conservative test (Spivak and Downey,
1998), it shows higher variability between
colonies (Espinosa-Montano et al., 2008). The
pin-killed brood assay is preferred in most Eu-
ropean breeding programs based on its higher
repeatability, correlation with a removal of
mite infested brood (Hoffmann, 1996), and
lower costs (Espinosa-Montano et al., 2008).
For the pin-killed brood test, usually 50
cells containing young pupae are pierced with
a fine insect pin. Since the removal of dam-
aged or killed pupae in the course of time fol-
lows a sigmoid function, the highest discrim-
inatory power of the test is reached when on
average 50% of the pupae are removed. There-
fore, the time interval between piercing the
cells and checking should be adapted to the
average removal response of the test popula-
tion. If the average removal rate is much lower
than 50%, prolonging the time interval will re-
sult in higher differences among colonies with
high and low hygienic behavior. If it is much
higher than 50%, a shorter time interval should
be realized in further test repetitions.
4.3. Grooming behavior
Auto- and allo-grooming are resistance
mechanisms against Va rro a in its original host
(reviewed by Boecking and Spivak, 1999;
Rath, 1999), but its quantitative contribution
to mite resistance in A. cerana is still not clear
and seems to be very limited. Grooming be-
havior has also been observed in A. mellifera
(Bozic and Valentincic, 1995; Thakur et al.,
1997), but compared to A. cerana, grooming
seems to be less effective in A. mellifera (Peng
et al., 1987; Büchler et al., 1992; Fries et al.,
1996).
Although it is affected by numerous bio-
logical and environmental factors, the propor-
tion of chewed mites in the debris of a colony
can be used as an indicator of grooming suc-
cess under field conditions (Moosbeckhofer,
1992; Büchler, 1993,2000; Rosenkranz et al.,
1997; Bienefeld et al., 1999; see also Rinderer
et al., 2010 for more details and references).
After several generations of selection in a
test population, colonies selected for this trait
showed significantly more damaged mites and
lower infestation rates compared to unselected
colonies (Büchler, 2000). However, the esti-
mated heritability was too low (h2<0.15;
Ehrhardt et al., 2007) to justify the laborious
sample collection and processing in a large-
scale selection program.
4.4. Postcapping stage duration
The postcapping stage duration (PSD) lim-
its the reproduction of Va r roa in sealed brood
cells. There is a median heritability but low
variability of the average PSD among Euro-
pean subspecies (Büchler and Drescher, 1990;
Breeding for resistance to Varroa destructor in Europe 7
Le Conte et al., 1994; Schousboe, 1986).
However, selection for faster development of
worker brood could be quite effective if re-
alized by direct selection on the reproductive
individuals (queens and drones; Moritz and
Jordan, 1992;LeConteetal.,1994; Siuda and
Wilde, 1996).
Wilde and Koeniger (1992) selected a line
of bees with a significantly shorter PSD com-
pared to A. m. carnica and A. m. caucasica
control colonies (276.4 hours versus 287.4
and 289.1 hours, respectively), but the ob-
served effects on the reproductive success
and the population increase of Va r ro a in
test colonies were not significant (Siuda and
Wilde, 1998). The selection program was
stopped after seven generations, recogniz-
ing that the achieved breeding progress of
0.2–4.2 hours per backcrossed generation re-
mained insignificant (Siuda et al., 1996).
4.5. Mite reproduction
The failure to reproduce in worker brood
is a basic aspect of the mite’s biology in A.
cerana, and thus it is a factor that enables co-
existence between Va r ro a and its native host.
Reproduction of Va r ro a in A. mellifera brood
has been intensively studied by numerous au-
thors (reviewed by Fries et al., 1994). How-
ever, the impact of bee genetics on the repro-
ductive success of Varro a is still unclear. The
significant lower fertility of Va r roa in African-
ized bees compared to European strains that
was observed in previous studies (Rosenkranz
and Engels, 1994; Rosenkranz, 1999) has not
been observed subseuqently (Garrido et al.,
2003), although the Africanized bee popula-
tion has managed to retain its high resistance
level to the mites.
Reduced mite fertility was observed in an
isolated apiary where the colonies were main-
tained without treatment and were selected for
low mite fertility over 9 years at the institute in
Lunz/Austria (Pechhacker et al., 1996). How-
ever, a comparison of bees and mites from
this isolated population to bees and mites from
Udine/Italy showed a significant effect on mite
fertility due to the origin of the mites and not
due to the origin of the bees (Milani et al.,
1999).
Recently, Rosenkranz et al. (2009)com-
pared the infertility rates of Va r roa in A. m.
carnica,A. m. ligustica,A. m. mellifera,SMR
(now called VSH; Rinderer et al., 2010), and
Gotland bees, and they did not detect signif-
icant effects of bee type or sex of the bee.
However, they found significant differences
in the pattern of cuticular compounds on the
bees, which should be further studied be-
cause certain compounds can reduce (Milani
et al., 2004)orstimulateVa r roa reproduc-
tion (Trouiller and Milani, 1999; Garrido and
Rosenkranz, 2004). Thus, selection for spe-
cific bee cuticular patterns might have the po-
tential to improve Va r ro a resistance in Euro-
pean bees.
5. BREEDING PROGRAMS
In most European countries, selection and
breeding activities are mainly realized by nu-
merous small-scale beekeepers with the sup-
port of several governmental institutions. In
most countries, specialized bee-breeding as-
sociations have been formed, and selection
guidelines have been compiled by beekeeper
associations and governmental authorities to
coordinate activities.
Most breeding programs use pure sub-
species and are oriented towards preserving
and improving local populations. Recently,
significant progress has been achieved by es-
tablishing a genetic evaluation of performance
test data, based on a BLUP animal model
adapted to the peculiarities of honey bee ge-
netics and reproduction (Bienefeld et al., 2007,
2008). This model estimates breeding values
for queen and worker effects based on sev-
eral colony traits, and it also considers envi-
ronmental effects. A central online database
with about 100 000 registered colonies of A.
m. carnica, A. m. ligustica, A. m. mellifera,and
A. m. siciliana comprising test data from Aus-
tria, Germany, Italy, Norway and Switzerland
has been established at the Länderinstitut für
Bienenkunde in Hohen Neuendorf, Germany
(http://www.beebreed.eu).
8 R. Büchler et al.
Within Europe, cross breeding of different
subspecies or strains is of minor importance;
it is used systematically in the selection of
Buckfast bees and occasionally also by single
breeders in search for new traits.
Meanwhile, resistance to Va r roa has been
recognized as a relevant selection criterion in
most European bee breeding programs. In the
following, we describe the AGT breeding pro-
gram as an example of an ambitious selec-
tion strategy based on pure populations, and
investigations of Russian Honey Bee (RHB)
lines from the US that received special atten-
tion as promising candidates for crossbreeding
efforts.
5.1. AGT selection strategy
The Arbeitsgemeinschaft Toleranzzucht
(AGT, http://www.toleranzzucht.de) selection
program was founded in 2003 to support the
selection and propagation of productive, gen-
tle queens with high resistance against Va r roa
and other diseases. Basically, the selection
program consists of three elements: (a) pre-
selection in a large population; (b) survival
testing of pre-selected breeder colonies; and
(c) drone selection under natural infestation
pressure.
5.1.1. Pre-selection in a large population
About 150 bee breeders are involved in test-
ing about 2000 queens per year. As a sustain-
able selection progress directly depends on the
size and the genetic variability of the popu-
lation (Moritz, 1986), the limits to the struc-
ture of bee breeding in Europe – namely small
numbers of colonies per apiary (uniform en-
vironmental conditions) and small numbers
of colonies per breeder (usually less than
100 colonies) – have been overcome by orga-
nizing a systematic exchange of test queens
among all breeders and apiaries within the
AGT. This approach guarantees optimal eval-
uation of environmental and genetic effects
in the central data processing. Using the esti-
mated breeding values, all tested queens can
be directly compared to each other, regardless
of their individual test environment.
The uniform protocol for the annual test-
ing is based on Apimondia recommendations
(Ruttner, 1972) and includes monitoring of the
Varro a population growth during the brood
season and repeated pin-killed brood tests for
brood hygiene behavior according to the meth-
ods described above. The central database at
the institute in Hohen Neuendorf has been
adapted accordingly, and breeding values for
Varro a resistance are calculated on the ba-
sis of mite population growth and pin-killed
brood test data in addition to breeding values
for honey productivity, gentleness, and swarm-
ing behavior. Work is ongoing to combine the
breeding values of all individual criteria into a
single selection index.
The AGT publishes an annual breeding reg-
ister with the breeding values of all tested
colonies, providing a comprehensive overview
over the whole population and stimulating the
exchange and propagation of valuable stock.
5.1.2. Survival test of pre-selected
breeder colonies
Garrido and Büchler (unpubl. data) ob-
served the wintering ability of untreated
colonies with regard to bee population size and
Varro a and virus infestation levels in differ-
ent locations in Germany. Colonies with in-
festation levels below 10% of the adult bees
and with adult bee populations of more than
10 000 bees in the fall had a high probability of
surviving the winter. The size of the bee popu-
lation in spring in relation to its size in October
(wintering index) proved to be a useful indica-
tor for the health status of the colony.
Within the AGT breeding program, bee
breeders are encouraged to refrain from us-
ing acaricides on test colonies with an infes-
tation of less than 1 mite/10 g bees in the sam-
ples routinely taken during the first decade of
July. Subsequently, these pre-selected colonies
are monitored for their mite infestation and
bee population development on a monthly ba-
sis. As soon as colonies come close to 10%
mite infestation or decrease to less than 10 000
bees, they are taken out of the test to pre-
vent domino effects by an invasion of mites
and secondary infections into the remaining
Breeding for resistance to Varroa destructor in Europe 9
population of untreated colonies. After the
spring evaluation, the wintering index will be
regarded together with the breeding values es-
timated from the preceding performance test to
finally select vital breeder colonies with a high
level of Varro a resistance.
5.1.3. Drone selection under natural
infestation pressure
The prime victim of parasitism by Va r -
roa mites is the drone population (Fuchs,
1990). Varro a infestation during the pupal
stage hampers the life expectancy and flight
abilities of drones (Schneider et al., 1988;
Duay et al., 2002; Bubalo et al., 2005), as well
as the development of spermatozoa (Schneider
and Drescher, 1987; Schneider et al., 1988;
Del Cacho et al., 1996;Duayetal.,2002,
2003).
Thus, Varro a infestation obviously has very
strong effects on the fitness of males in the
colonies. Büchler et al. (2006) studied the ef-
fect of different levels of Varro a infestation
on male fitness of 26 closely related drone
colonies in an isolated mating station. From
May to July 2005, brood activity, adult bee and
drone populations, and Va rro a infestation were
estimated in regular intervals. At the same
time, the mating success of the drone colonies
was checked using molecular paternity tests.
Using five closely linked microsatellite loci,
the genotypes of 412 workers from 48 queens
that were mated during this period could be as-
signed to one of the 27 haplotypes identified
in the drone colonies. The Va r roa infestation
level (range: 0.2–8.8 mites/10 g bees) proved
to be negatively correlated to the colony size
(range: 20–31 comb streets of bees), the num-
ber of adult drones (range: 2275–8725), the
mating success per drone (range: 2.7–18), and
the number of descendants per mating (range:
4–39; Fig. 2).
Fostered by the long distance of mat-
ing flights, the strong and direct competition
among drones from many different colonies
within the range of each drone congregation
area, and the multiple mating of each queen,
differences in the mite infestation of colonies
must result in strong selection effects under
Figure 2. Effects of Va r ro a infestation on compo-
nents of drone fitness. Significant correlations are
given in bold letters (* P<0.05; ** P<0.01).
natural mating conditions. This hypothesis is
also supported by the observation of a remark-
ably fast development of significant mite re-
sistance in some untreated A. mellifera popu-
lations.
Since 2004, the AGT breeding program is
taking advantage of this element of natural se-
lection by maintaining several isolated mating
areas (so called “tolerance mating stations”)
with untreated drone colonies (Büchler et al.,
2009). These drone donors usually descend
from several unrelated breeder queens in or-
der to achieve high genetic variablity within
the selected drone population.
As long as the mite infestation of a
given colony does not exceed the threshold
level (10 mites/10 g bees), the colonies are
maintained without any chemical treatments.
The sole remedy permitted for use to reduce
the mite load in these colonies at the end of
the mating season is the complete removal
of the sealed brood combined with introduc-
tion of a trapping comb (open brood comb
into the broodless colony which is removed af-
ter sealing). The anticipated losses of infested
drones are compensated for by a high number
of drone colonies (usually 30–100 colonies per
mating station).
5.2. Potential of crossbreeding local
stocks with comparatively resistant
stocks from far eastern Russia
To speed up selection efforts for V. destruc-
tor resistance, crossbreeding of comparatively
resistant stocks was envisioned as an addi-
tional option and alternative to selection that
10 R. Büchler et al.
is solely based on local populations. Promising
reports from the United States indicated a high
level of resistance to V. destructor in honey
bees from far eastern Russia (Rinderer et al.,
2001,2010). To test their potential for resis-
tance breeding, comparative investigations on
mite development and colony performance be-
tween Russian honey bee (RHB) from the US
and local A. m. carnica (C) were carried out in
Germany.
In a preliminary three-month study, a dis-
tinctly slower Varro a population increase in
RHB colonies from the USDA selection pro-
gram could be confirmed (Koeniger et al.,
2000). Subsequent large-scale studies by sev-
eral institutions using hybrids of RHB with
other strains from the US also confirmed the
slower increase of the Var roa population in
the RHB, but the interpretation of the re-
sults was ambiguous. In some experiments,
the colony performance of RHB was sig-
nificantly worse compared to C, which was
hypothesized as one main reason of slower
mite growth (Rosenkranz and Liebig, 2003;
Schuster, 2003). In other studies, the slower
mite growth could not be explained by poor
colony development (Berg et al., 2003).
To exclude possible hybrid effects, the
following experiment compared 12 different
RHB lines headed by purely mated queens
(n =113; Rinderer et al., ?) with three lo-
cal C lines (n =36; Berg et al., 2004). The
colonies were maintained without interfering
in colony performance and V. destructor pop-
ulation growth. After 1.5 years, the mite pop-
ulation (estimated from bee samples) of RHB
was 50% of that in C, and the colony strength
(estimated as number of occupied combs in
regular intervals) did not differ between the
two groups (Berg et al., 2005). The compar-
atively lower mite population increase in RHB
colonies could have been due to their signif-
icantly stronger hygienic behavior (based on
the pin-killed brood test) and a trend of hav-
ing a higher proportion of infertile mites in
their worker brood (estimated in at least 30
single infested brood cells/colony). However,
differences in the distribution of the mites on
adult bees and in worker brood as reported by
Rinderer et al. (2001) were not observed in this
experiment.
Although the mite population growth was
slower in the RHB colonies, it did increase
continuously and the colonies harbored an av-
erage of 10 000 mites/colony at the end of the
experiment. In addition, RHB showed unfa-
vorable attributes regarding their productivity
and gentleness, barring further thoughts of in-
cluding them into crossbreeding programs.
6. EFFICIENCY REVIEW
OF EUROPEAN SELECTION
ON MITE RESISTANCE
6.1. Survivability of selected and
unselected European honeybee
from different origins
To survey the success of selection programs
for Varro a resistance, 14 European strains of
bees were compared on an isolated Adriatic is-
land (Unije, Croatia) over a period of 2.5 years
(Berg et al., 2001; Büchler et al., 2002). Seven
strains originated from selection programs for
resistance to V. destructor (Carniolan n =5;
Buckfast n =1; hybrid n =1), and the
other seven were unselected strains (Carniolan
n=5; Buckfast n =1, Ligustica n =1). The
117 colonies were started as artificial swarms
(1.6 kg) with 270 Va r ro a each and were main-
tained according to normal management prac-
tices but without any treatment against Varr o a .
At the end of the experiment most of the
colonies had died, with only 15 colonies still
alive. While in the first year colony losses
mainly were attributed to difficulties in adap-
tation to the specific local conditions on the
island (hot and dry summer, strong winds),
colony losses in 2001 and 2002 were predom-
inantly caused by varroosis.
There were distinct differences in the sur-
vivability of the different strains of bees
(ANOVA, P<0.05), with the colonies orig-
inating from selection programs showing a
significantly higher proportion of surviving
colonies (11 out of 63 colonies) compared
to the unselected ones (4 out of 54 colonies;
Wilcoxon, P<0.01). However, the higher sur-
vivability rate of colonies from selected strains
could not be correlated to differences in the
relative natural mite mortality (mite mortal-
ity/1000 bees), the number of damaged mites
Breeding for resistance to Varroa destructor in Europe 11
in the natural mite fall, hygienic behavior (pin-
killed brood test), or the infertility rate of Va r -
roa in worker brood. Nevertheless, from the
second year on, the selected strains had sig-
nificantly stronger colonies compared to the
unselected strains (all Wilcoxon, P<0.05).
The higher colony strength and better survival
rate convincingly demonstrate the advantages
of the strains selected for Va r roa resistance,
compared to the unselected strains.
6.2. COLOSS genotype – environment
interaction test
Recently, a European-wide experiment was
initiated as part of the COLOSS project (http://
coloss.org) to estimate the resistance and vi-
tality of different European bee strains and
the interactions of these factors with different
environmental conditions. It comprises about
600 colonies of 17 different origins (A. m.
carnica, A. m ligustica, A. m macedonica, A.
m mellifera, A. m sicula)in26testapiaries
throughout Europe. The survival, bee popula-
tion and Va r ro a infestation development of the
colonies will be tested without using any aca-
ricides over several years.
7. PERSPECTIVES
High survival rates in some local
populations and significant variability in
the mite infestation levels between breeding
lines convincingly demonstrate there is poten-
tial to establish Va r ro a resistance in European
Apis mellifera populations. Several selection
tools suitable for use by beekeepers have been
developed and have been implemented in field
selection programs. However, the resistance
mechanisms are complex and are still only
partially understood. Furthermore, resistance
does not occur as an isolated interaction
between a host colony and its parasite, but de-
pends on hive management and environmental
conditions, including other pathogens.
Based on this current status we envision the
following perspectives for bee breeding in the
future:
•So far, only minor attention has been paid
to the importance of specific ecological
adaptations and the natural biodiver-
sity of honeybees for disease resistance.
Consequently, higher priority should be
placed on the investigation of genotype-
environment (climate, resources, manage-
ment) interactions and the development of
effective strategies to conserve and utilize
natural honeybee gene resources. This ef-
fort may also include changes in currently
used breeding techniques to boost natu-
ral selection mechanisms and high intra-
colony diversity.
•More research is needed to elucidate the
relevance and causality as well as the
molecular processes and the genetics of
resistance characters. Numerous questions
still need to be answered. For example:
How do bees influence the reproductive
success of Va rro a using chemical stimuli
for example? What role does the entomb-
ing behavior of infested pupae, known as
an important mechanism of resistance in A.
cerana,playinA. mellifera? Is there vari-
ability in susceptibility to virus infections
that influence susceptibility to Va r ro a ?
•Based on a better understanding of re-
sistance mechanisms, suitable selection
methods need to be developed and im-
proved. Identifying the genes involved in
Varro a resistance and establishing genetic
markers for resistance traits are very stim-
ulating challenges to incorporate charac-
ters such as grooming behavior that are
difficult to estimate under field conditions
and to speed up the selection progress.
The honeybee genome sequence as molec-
ular and genomic tool may enable such
projects, as Va rro a resistant bee popula-
tions become available.
•Accustomed management techniques have
to be revised. Regular and uniform treat-
ments of bee populations with highly ef-
fective acaricides are in opposition to field
selection for resistance. To support the
spread of more resistant stock, beekeep-
ers need to identify (through monitoring
infestation level) and exclude highly sus-
ceptible colonies from further propagation.
As soon as the individual infestation of
a colony exceeds certain threshold lev-
els colonies should either be destroyed, or
12 R. Büchler et al.
treated and requeened to prevent domino
effects. Preference of shorter brood rearing
periods, acceptance of temporary breaks
in brood rearing and complete brood re-
moval once a season are some tools bee-
keepers can use to lower the population
growth of Varro a and thus to reduce their
dependence on the use of miticide treat-
ments which mask the advantages of mite
resistant stock.
ACKNOWLEDGEMENTS
We like to thank Marina Meixner for her helpful
comments on the manuscript.
Sélection d’abeilles résistantes à Varroa destruc-
tor en Europe.
résistance au Varroa /programme d’élevage /ca-
ractère tolérant /vitalité /sélection naturelle
Zusammenfassung –Auslese auf Widerstandsfä-
higkeit gegen Varroa destructor in Europa. Seit
Einzug der Va rro a Milbe in Europa sind in einigen
Regionen Völker aufgetreten, die langfristig oh-
ne Behandlungsmaßnahmen überleben. Eine wei-
tergehende Untersuchung von 82 derartiger Völ-
ker (VSB) in Frankreich zeigte eine mittlere
Überlebensdauer von 7,8 Jahren. Der Va rro a -
Befallsanstieg während der Brutsaison verlief signi-
fikant schwächer als bei normal anfälligen Kontroll-
völkern (Abb. 1). Dabei spielen offenbar ein inten-
sives Putzabwehr- und Bruthygieneverhalten eine
Rolle, möglicherweise aber auch häufige Schwar-
mereignisse, spezifische Umweltbedingungen, Ver-
änderungen der Milbenpopulation und die Vi-
rusprävalenz.
Durch einen völligen Verzicht auf Bekämpfungs-
maßnahmen ist es innerhalb weniger Generatio-
nen sowohl in einem kommerziellen Zuchtbetrieb
in Frankreich als auch in einer isolierten Versuchs-
population in Schweden zur Auslese resistenter
Bienen gekommen. Als mögliche Ursachen der ab-
geschwächten Befallsentwicklung wurden u.a. eine
geringere Brutdynamik und ein geringer Brutbefall
bezogen auf die Gesamtmilbenzahl beobachtet.
Aufgrund umfangreicher Untersuchungen liegen
heute standardisierte Methoden zur Beurteilung der
Befallsentwicklung und der Bruthygiene vor, die
dank guter Heritabilitäten und effizienter Zucht-
wertschätzverfahren in vielen europäischen Län-
dern Eingang in die Zuchtpraxis gefunden haben.
Zuchtprogramme zur Selektion auf Putzabwehr-
verhalten, kürzere Verdecklungsdauer oder beein-
trächtigte Var roa-Reproduktion scheiterten hinge-
gen an ineffizienten Messverfahren, geringer gene-
tischer Variabilität oder unzureichend geklärter bio-
logischer Zusammenhänge.
Beispielhaft wird die Organisation eines umfangrei-
chen Zuchtprogramms zur Steigerung der Resistenz
leistungsfähiger Zuchtlinien vorgestellt. Die Ko-
operation zahlreicher Zuchtbetriebe, einheitliche
Prüfstandards und eine gemeinsame Zuchtwert-
schätzung ermöglichen eine Auslese in großer
Population. Potenzielle Zuchtvölker werden auf
ihre Überwinterungsfähigkeit ohne vorangehende
Behandlung gegen Va rro a geprüft und durch den
Betrieb von Belegstellen mit unbehandelten Droh-
nenvölkern werden Effekte unterschiedlicher Va r -
roa-Anfälligkeit auf den Paarungserfolg von Droh-
nen (Abb. 2) in die Selektion einbezogen.
Aufgrund schlechterer Honigleistung und Sanftmut
konnten US-Linien Russischer Bienen (RHB) trotz
relativ hoher Widerstandsfähigkeit in Europa kei-
ne Bedeutung erlangen. Die meisten europäischen
Zuchtprogramme zielen auf einer stetige Verbes-
serung einheimischer Populationen. Inwieweit es
auf diesem Weg möglich ist, hochgradig resisten-
te und zugleich leistungsfähige Zuchtlinien zu ent-
wickeln, bleibt offen. Immerhin zeigte sich in einem
mehrjährigen Vergleichsversuch eine höhere Über-
lebensfähigkeit der bereits auf Resistenz gegen Va r-
roa ausgelesenen Linien.
Wichtige Zukunftsperspektiven ergeben sich aus
der Weiterentwicklung der Selektionsmethoden, der
Berücksichtigung spezifischer Umweltanpassungen
und verbesserter Haltungstechniken.
Varroa Resistenz /Zuchtprogramm /Toleranz-
merkmal /Vitalität /natürliche Selektion
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