honey bee colonies from different races show variation in defenses against the varroa mite in a...
TRANSCRIPT
Honey bee colonies from different races show variationin defenses against the varroa mite in a ‘commongarden’Meral Kence1, Devrim Oskay2, Tugrul Giray3* & Aykut Kence11Department of Biology, Middle East Technical University, 06800 Ankara, Turkey, 2Department of Agricultural
Biotechnology, Namık Kemal University, 59030 Tekirdag, Turkey, and 3Department of Biology, University of Puerto Rico,
PO Box 23360, San Juan, PR 00931, USA
Accepted: 27 June 2013
Key words: mite-biting behavior, hygienic behavior, Apis mellifera, Apis mellifera caucasica, Apis
mellifera syriaca, Apis mellifera anatoliaca,Apis mellifera carnica, Hymenoptera, Apidae, Acari,
Varroidae
Abstract Honey bee [Apis mellifera L. (Hymenoptera: Apidae)] genetic diversity may be the key to responding
to novel health challenges faced by this important pollinator. In this study, we first compared colo-
nies of four honey bee races, A. m. anatoliaca, A. m. carnica, A. m. caucasica, and A. m. syriaca
from Turkey, with respect to honey storage, bee population size, and defenses against varroa. The
mite Varroa destructor Anderson & Trueman (Acari: Varroidae) is an important pest of honey bee
colonies. There are genetic correlates with two main defenses of bees against this parasite: hygienic
behavior, or removing infested brood, and grooming, which involves shaking and swiping off mites
and biting them. In the second part of this study, we examined the relationship of these two types of
defenses, hygiene and grooming, and their correlation with infestation rates in 32 genetically diverse
colonies in a ‘common garden’ apiary. Mite biting was found to be negatively correlated with mite
infestation levels.
Introduction
Honey bees, Apis mellifera L. (Hymenoptera: Apidae), are
important as generalist pollinators both for conserving
wild flora and for increasing agricultural crop yields
(Morse & Calderone, 2000). Recent losses of honey bee
colonies across the world (Oldroyd, 2007; vanEngelsdorp
et al., 2008, 2011; Giray et al., 2010; Neumann & Carreck,
2010) has raised the question of synergistic effects ofmulti-
ple factors being responsible for these losses (see Huang &
Giray, 2012).
Understanding defenses of honey bees against key
enemies, such as the ectoparasitic mite Varroa destructor
Anderson & Trueman (Acari: Varroidae), could help miti-
gate multiple problems such as virus transmission, honey
contamination with pesticides, and the ectoparasite itself.
Various behavioral defense mechanisms against varroa
have been observed in honey bees, which are targeted
against the mite on brood or on adult bees. Hygienic
behavior is a heritable trait that involves the ability of
bees to detect, uncap, and remove mite-infested brood
(Vandame et al., 2000; Harbo & Harris, 2005; Ibrahim &
Spivak, 2006). Initiation of hygienic behavior is related to
genes involved in the workers’ sensitivity to odors released
by dead or damaged brood (Swanson et al., 2009). Mite
grooming has recently been shown to differ across African-
ized and European bees, Russian bees, and two selected
lines in the new world (high and low resistance). A com-
parison of 4–9 colonies of each race and line showed that
these differences were correlated with the mite infestation
levels of the colonies (Guzman-Novoa et al., 2012). Mite-
grooming behavior has been described as the ability of
adult bees to injure and remove Varroa mites from
their bodies (Peng et al., 1987; Arechavaleta-Velasco &
Guzman-Novoa, 2001). Biting behavior is the ability
of adult bees to catch and bite Varroa mites with their
mandibles, rendering them ineffective (Aumier, 2001;
Rivera-Marchand et al., 2012). Removal and biting could
be distinct components of grooming, because shaking and
*Correspondence: Tugrul Giray, Department of Biology, University
of Puerto Rico, PO Box 23360, San Juan, PR 00931, USA.
E-mail: [email protected]
© 2013 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 1–8, 2013 1
DOI: 10.1111/eea.12109
swiping off the mite are not always associated with mite
biting (see Guzman-Novoa et al., 2012; Rivera-Marchand
et al., 2012); moreover, assays have shown that mites that
are on a substrate and not on bees are also attacked and bit-
ten by Africanized bees (e.g., Rivera-Marchand et al.,
2012). In addition, in several studies under controlled con-
ditions, injured mites found on the hive floor were not
always correlated with shaking and swiping behavior
(reviewed inGuzman-Novoaet al., 2012).
Differences in these defenses across genetically different
bees found in native habitats could be an important
resource for a resilient agricultural bee population. In Tur-
key, there are several genetically distinct races of bees
within a relatively small area (Kandemir et al., 2000;
Bodur et al., 2007; Solorzano et al., 2009). Turkey extends
over the Anatolian peninsula, which was an important ref-
uge for animals and plants during the Pleistocene glacia-
tion (Kosswig, 1955; Hewitt, 1999). The role of Anatolia as
a refuge is thought to explain its large biological diversity,
which is equal to the entire European continent in terms
of numbers of species (Davis, 1965–1985; Kence, 1987;Davis et al., 1988; M�edail & Qu�ezel, 1999; G€uner et al.,
2000). For honey bees, the situation is similar to other spe-
cies, in that based on morphology and geographic distri-
bution, five honey bee races have been identified in Turkey
(Ruttner, 1988; Kandemir et al., 2000). This represents
about 20% of the racial diversity in the world. Ruttner’s
hypothesis that these bees are from the same lineage fits
with mitochondrial DNA analyses results (Kandemir
et al., 2006) and genomic variation studies (Kandemir
et al., 2000, 2005, 2006;Whitfield et al., 2006; Bodur et al.,
2007).
In this study, we first compared the levels of defense and
infestation, as well as honey production and colony
population growth, among four subspecies of honey bees,
A. m. anatoliaca (from D€uzce, Yı�gılca, and Mu�gla prov-
inces),A. m. caucasica (fromArtvin),A. m. carnica (from
Kırklareli), and A. m. syriaca (from Hatay), under the
same environmental conditions in a ‘common garden’
apiary (Figure 1). Common garden is a technique whereby
organisms from various populations are brought together
in one location to examine genetic differences rather than
the differences between environments from which the
populations originate (e.g., Wikelski et al., 2003). We
hypothesized that there would be differences in multiple
traits across races. We predicted that A. m. syriaca, the
southern race from subtropical areas, may have greater
disease protection, whereas northern races may have
greater honey storage, with lower brood production
(Rivera-Marchand et al., 2008). In subtropical regions,
bees can find floral resources to collect nectar and pollen
for longer periods of time than in northern and central
regions and brood rearing coincides with the duration of
floral availability. We also examined the relationship
between hygienic behavior and grooming as potential
defenses that may influence Varroa infestation rates in
colonies. We tested the hypothesis that infestation would
be smaller in colonies with a high level ofVarroa defenses.
Materials and methods
Colony assignment to one of the four races was confirmed
based on multivariate morphometric analysis (Kandemir
et al., 2000) and genetic analysis based on microsatellite
variation (Bodur et al., 2007; Ivgin-Tunca, 2009). Colo-
nies were transported from their original locations to the
study site at theMiddle East Technical University, Ankara,
Turkey, i.e., over a distance of ca. 240 (Duzce) to
Figure 1 Colonies from populations of fourApis mellifera races were brought from their original locations near the borders of Turkey with
Georgia, Bulgaria, and Syria, and from along the Aegean and Black Sea coast to the ‘common garden’ in the central province Ankara.
Numbers indicate the original locations ofA. m. caucasica (1),A. m. carnica (2),A. m. syriaca (3), andA. m. anatoliaca (4a, b), and the
study site (5).
2 Kence et al.
1 000 km (Artvin) in the summer of 2009 (Figure 1). All
colonies were kept according to standard methods and in
the same apiary. Colonies were standardized for initial
population size at the start of the study to 10 frames cov-
ered with bees with similar brood population, honey, and
pollen. To ensure that we only studied newVarroa infesta-
tions, all the colonies were treated for Varroa in the fall of
2009.We found nomites on sticky traps placed in colonies
on the last 2 days of the treatment. We used the acaricide
Flumethrin (Varostop; Lavita, Istanbul, Turkey), following
manufacturer’s instructions. Colonies overwintered and
were monitored through spring and summer of 2010. We
examined honey storage (number of frames with stored
honey), brood (number of frames as percentage of frame
area containing cells with eggs, larvae, and pupae), and ini-
tial and final adult bee population size measured as num-
ber of frames covered with bees to the nearest quarter of a
frame (Giray et al., 1999, 2000; Rivera-Marchand et al.,
2012).
In summer, 8 months after the acaricide treatment, we
measured Varroa infestation level (% infested cells) in col-
onies by examining 150 (if infestation >5%) to 300 (<5%)
sealed brood cells (modified from Fries & Bommarco,
2007; Guzman et al., 2007). Bees develop from individual
eggs laid by the queen in wax cells. These cells are sealed
when the larvae are ready to pupate. Any infesting Varroa
mites are trapped inside the cells until the cell cap is
opened. Regression of the generally low infestation rates to
final colony population, measured as number of frames
covered with bees, was marginally non-significant
(r = 0.42, P = 0.07; n = 24). In all further correlation
analyses on infestation rates, effect of final colony popula-
tion was statistically controlled by use of residuals from
the regression of infestation rate on colony population.
We examined hygienic behavior in each colony (except
two that were excluded accidentally) using the freeze-killed
brood assay technique (Spivak & Reuter, 2001; Ibrahim &
Spivak, 2006). A piece of brood comb with ca. 150 sealed
cells was cut and frozen overnight at�20 °C. The next day,after thawing, the piece was put back in the original comb
and placed in the brood nest of the test colony (Spivak &
Downey, 1998). After 24 and 48 h, cells that were
completely cleaned were counted. The level of hygienic
behavior was expressed as the percentage of cleaned cells
relative to the total number of frozen cells. Hygienic
behavior is generally measured over time and in two inde-
pendent tests when single colony phenotype is important,
for instance in selection studies. This is especially impor-
tant when all colonies cannot be tested simultaneously
under similar environmental (nectar availability, weather)
and colony conditions (Varroa infestation level). In this
study, we standardized initial Varroa infestation levels and
measured all colonies at the same time and place (similar
to Rivera-Marchand et al., 2008, 2012).
Mite biting by bees and grooming success were exam-
ined in 10 assays on groups of 10 bees from 10 colonies
chosen from the various races (three each of A. m. cauca-
sica and A. m. syriaca, two each of A. m. anatoliaca and
A. m. carnica). Assays were performed in plastic petri
dishes of ca. 150 ml volume (Aumier, 2001; Rivera-
Marchand, 2006). A detailed description of the method
and video taping of mite biting and mite removal is pre-
sented elsewhere (Rivera-Marchand et al., 2012). Briefly,
mites were collected from heavily infested additional colo-
nies in the apiary. Mites were kept in a petri dish equipped
with a moistened paper wick to provide humidity at ambi-
ent temperature (28 °C).Worker beeswere collected into a
plastic cup from the brood nest of 10 experimental colonies
and kept in flight cages in the same roomas themites. Indi-
vidual petri dishes (9 cm in diameter) were prepared as
assay arenas: their plastic lids were perforated with needle-
size air holes, and one introduction hole large enough for
individual bees. Bees were captured by allowing them to
walk into a small vial, then the vial was placed over the
introduction hole, and the bees walked into the petri dish.
The introduction hole was covered with a clear tape. This
procedure was repeated until 10 individuals were placed in
the arena. Bees were given 5 min to become accustomed
to the petri dish.
The tape covering the introduction hole was lifted
slightly to place a single mite on the clear floor of the assay
arena, using a fine brush. For contrast, the petri dishes
were kept on a white laboratory bench. A stopwatch was
started after the mite made the first contact with a bee in
the arena. The interactions were observed for 2 min. Shak-
ing and swiping or biting behavior exhibited by bees in
contact with the mite were recorded during this time
(Rivera-Marchand et al., 2012). Vigorous shaking and
swiping has been termed ‘intense grooming’ in previous
studies (Guzman-Novoa et al., 2012; Rivera-Marchand
et al., 2012), here we prefer ‘shaking and swiping’ to
emphasize that ‘biting’ is a distinct behavior. Two observ-
ers simultaneously recorded the behaviors for each assay
and in case of disagreement in numbers of events, the low-
est common number was recorded. Bees were tested in
groups of 10 with one focal bee paint-marked on the tho-
rax. Per colony, the behavior of individual focal bees was
recorded (i.e., biting, shaking, or swiping after first con-
tact, or no response). For each colony, the percent of focal
individuals that responded by grooming behavior was
used in analyses to correlate with mite infestation levels in
the colony. Percentages for various colonies of a single race
were averaged and this average was reported as race per-
centage in the results.
Bee defenses againstVarroa 3
Although we aimed for 10 colonies per race, due to dif-
ferent external conditions we started with sevenA. m. car-
nica, eight A. m. caucasica, eight A. m. syriaca, and 11
A. m. anatoliaca colonies. We obtained population and
colony condition measurements for all 34 colonies; how-
ever, in the hygiene test two colonies (A. m. anatoliaca)
were excluded bymistake.
Statistical analysis
ANOVA was performed to determine any racial differ-
ences in the parameters measured (Sokal & Rohlf, 1995).
Correlation analysis on residuals for percent infestations
was applied to identify associations of mite infestation lev-
els and defense behaviors. In addition, a one-way Kruskal–Wallis test was used to further analyze the relationship of
biting behavior and infestation rate, comparing the five
colonies without individuals biting mites to the five with
biting individuals. These analyses were performed using
the statistical package JMP v6 (SAS Institute, Cary, NC,
USA). We also tested whether the behavioral traits could
be used to identify colonies from the four subspecies using
canonical variate analysis (NTSYSpc 2.20e; see supporting
material, Table S1 and Figure S1).
Results
The four bee races appear to differ with respect to various
(combinations of) characteristics, such as final honey
stores, brood, adult population size, Varroa infestation,
and hygienic behavior (Figure 2). Apis m. syriaca colonies
combine small honey stores and large brood area, whereas
A. m. carnica colonies have large honey stores, large
brood, and high bee numbers. Apis m. caucasica colonies
have large honey stores but small brood areas. Apis
m. anatoliaca are in between, yet more similar to
A. m. caucasica especially at higher Varroa infestation
levels. A discriminant analysis based on the combination
of these three measures with infestation rate and hygienic
behavior, separates the colonies of all subspecies
except A. m. anatoliaca, of which some colonies are
distributed across the other subspecies (Figure 1, Figure
S1, Table S1).
Varroa infestation rates were lower than 10% for all col-
onies at the start of measurements in the summer, indicat-
ing that the initial control of mite populations with the fall
acaricide application was sufficient to maintain Varroa
infestation levels below the critical level for a new acaricide
A B C
D E F
Figure 2 Comparison of colonies from fourApis mellifera races,A. m. caucasica,A. m. carnica,A. m. syriaca, and A. m. anatoliaca.
Mean (+ SE) (A) honey stores (no. frames with honey) (F3,33 = 7.761), (B) brood (no. frames filled with brood) (F3,33 = 2.87), (C) adult
population (no. frames covered with bees) (F3,33 = 4.653), (D)mite infestation rate (% infested brood) (F3,33 = 3.04), (E, F) hygiene, i.e.,
removal of frozen brood (E) after 24 h (F3,31 = 4.021), and (F) after 48 h (F3,31 = 5.089; all P<0.05). For eachmeasure, overall ANOVA
indicated a significant difference across races. Numbers in bars indicate the number of colonies per race. Means capped with different
letters are significantly different (Scheff�e’s post-hoc comparisons: P<0.05).
4 Kence et al.
treatment. Yet, a statistically significant difference across
races was found (ANOVA: P<0.05; Figure 2D).
A colony can be identified as hygienic based on a prede-
termined cutoff such as >80% or 95% removal of freeze-
killed brood after 48 h [e.g., Rivera-Marchand et al.
(2008) and Ibrahim & Spivak (2006), respectively]. As per
either criterion, similar results were obtained for hygiene
level of the four races (see Figure 2F). Quantitative differ-
ences in hygienic behavior after 48 and 24 h were similar,
although only two A. m. carnica colonies reached 80–100% removal rate at 24 h (Figures 2E and 3A).
Based on 32 colonies, overall hygienic behavior score
and mite infestation levels were negatively, but not signifi-
cantly, correlated (Figure 3A). The negative correlation of
infestation level and mite-biting behavior was found to be
significant, despite the limited sample size of 10 colonies
(Figure 3B). Themean (� SE) infestation levels of the col-
ony groups with vs. without mite biting were found to be
significantly (0.88 � 0.73 vs. 6.26 � 1.9%, respectively;
one-way Kruskal–Wallis test: v2 = 3.76, d.f. = 1, P<0.05).Colonies where biting behavior was observed were
distributed across the races tested: one each in
A. m. caucasica, A. m. carnica, and A. m. anatoliaca, and
two colonies of A. m. syriaca. Shaking and swiping was
not seen in A. m. anatoliaca bees (200 bees tested, from
two colonies), whereas it was observed at a frequency of
16.7% in A. m. syriaca and A. m. carnica colonies (both
races: 300 bees tested, from three colonies), and 30% in
A. m. caucasica (200 bees tested, from two colonies).
These percentages of shaking and swiping for mite
removal for the 10 colonies were not found to correlate
with Varroa infestation levels, when analyzed indepen-
dently of biting behavior (R = 0.08, P>0.8; n = 10).
Partial correlations were calculated across levels of
defense and residual infestation (controlling for colony
population) for 10 colonies for which all the measure-
ments were complete. The negative correlation of defen-
sive mite biting with Varroa infestation was highly
significant, whereas that of hygienic behavior with mite
infestation was not significant (Table 1). Interestingly,
shaking and swiping showed a significant positive correla-
tion with residuals of infestation rate, when correlation
with mite biting was controlled in the partial correlation
analysis.
Discussion
The most important finding of this study is the difference
in Varroa infestation levels, hygienic behavior, and other
population characteristics among races of honey bees
under similar environmental conditions. Moreover, one
type of grooming, mite-biting behavior has a stronger neg-
ative correlation with Varroa infestation levels than the
hygienic behavior in a subset of these genetically diverse
colonies. The highest levels of hygienic behavior and the
lowest infestation levels in the commercially less desirable
southern bee, A. m. syriaca, fit the hypothesis that (sub)
tropical bees have greater defenses against parasites.
Finding variation in Varroa defenses among natural
populations in Turkey is in concordance with the discov-
ery of honey bee populations surviving without chemical
A
B
Figure 3 Correlation ofVarroa infestation rate (% bee larvae
found withVarroamites) andApis mellifera hygiene as defense
against the parasitic mite (% focal bee individuals that responded
by grooming behavior). (A)Mite infestation vs. bee hygienic
behavior after 24 h. The correlation of hygienic behavior and
residuals of infestation rate (controlled for colony population)
was not significant (R = �0.28, 0.05<P<0.1; n = 32). (B)Mite
infestation vs. mite biting by bees (R = �0.68, P<0.05; n = 10).
Table 1 Partial correlations across defenses and residuals for
infestation level of 10 colonies of honey bees
% infestation Grooming Biting
Hygiene �0.20 �0.68* �0.04
Biting �0.82* 0.59
Shaking and swiping 0.74*
*0.05<P<0.01.
Bee defenses againstVarroa 5
treatments, or uninfested with mites in Europe and
elsewhere (Fries & Bommarco, 2007; Le Conte et al.,
2007). That this variation is exhibited under common-gar-
den conditions supports genetic differences in ectoparasite
defenses. It has been reported that the tropical Africanized
honey bee, Apis mellifera scutellata Lepeletier, deals with
mites more effectively than European bees, with hygienic
behavior and other behaviors such as grooming, increased
swarming, and absconding contributing to the difference
(Frazier et al., 2010).
In a recent study of grooming in Africanized, European,
and Russian bees and two selected lines [low (SL) and high
(SH) varroa population lines of Russian bees], intense
grooming (shaking and swiping) was correlated with lower
mite infestation levels in colonies; however, biting was not
directly examined (Guzman-Novoa et al., 2012). In this
study, shaking and swiping were not found to be corre-
lated with infestation level when taken independently of
biting behavior. An important methodological difference
between the two studies is in the introduction ofmites into
the assay arena. Guzman-Novoa and colleagues intro-
duced the mite on the dorsal thorax of a bee. In previous
studies we found that this method precludes the assess-
ment of biting, and biases the test toward shaking and
swiping. In this study, we see that placing the mite on the
arena floor biases the test toward biting behavior, and also
makes it easier for the bees to shake and swipe off the mite.
In many instances, mites were not able to reach the dor-
sum of the thorax of the bee (see also Rivera-Marchand,
2006; Rivera-Marchand et al., 2012). The observation that
shaking and swiping behavior is higher in colonies with
higher infestation rates may indicate the induction of this
behavior. The link between biting and shaking/swiping
behaviors should be examined further, as it could help us
develop new selection targets to develop Varroa-resistant
bees for beekeepers.
Genetic diversity in honey bee colonies has been shown
to increase colony fitness (Jones et al., 2004;Mattila & See-
ley, 2007; Mattila et al., 2008) and to raise the level of
hygienic behavior efficiency. Honey bee populations in
Turkey are found to genetically vary more than European
but less than African bees (Bodur et al., 2007; Ivgin-Tunca,
2009). However, the past thinking that a correlated set of
traits resulting in behavioral syndromes (Giray et al., 1999,
2000 – for behavioral development and defense; Hunt,
2007; Tsuruda & Page, 2009 for defensiveness and related
traits) could havemade any desiredmite resistancemecha-
nism inaccessible for commercial bees. Therefore, the
apparent independence of honey storage, Varroa defenses,
and colony population traits found in the colonies from
these four honey bee races highlight the potential for com-
bining desirable traits for resilient and productive bees.
Acknowledgments
We acknowledge support from the European Union
COST program through a grant to AK administered by
the Turkish Scientific and Technical Research Council
(TUBITAK) and the USDA-NIFA grant (2009-05291)
to TG. We also thank members of the A. Kence, M.
Kence, and T. Giray laboratories for careful reading
and feedback on previous versions of this manuscript.
Comments by two anonymous reviewers improved the
manuscript. Special thanks go to TEMA foundation,
Mr. Ali Nihat Gokyigit, and D€uzce, Hatay, Kırklareli,
Mu�gla provincial Beekeepers Association directors,
and members for supplying us with local bees used in
establishing the common-garden apiary.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Discriminant analysis combining data on
honey stores, brood, adult population size, Varroa infesta-
tion rates, and hygienic behavior for four Apis mellifera
races. Eigenvalues of canonical variation analysis and
statistics are presented in Table S1. Three races are well
separated: A. m. syriaca, A. m. caucasica, and A. m. car-
nica. The colonies of A. m. anatoliaca fell between the
other races.
Table S1. Canonical variation analysis (CVA) function
eigenvalues and explained variance (Wilk’s lambda
= 0.144; F15,44.6 = 3.030, P = 0.003) for separation of four
Apis mellifera races based on behavioral and colony
measures.
8 Kence et al.