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: tgiray2@yahoo.com

© 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.