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Behav Genet (2017) 47:335–344
DOI 10.1007/s10519-017-9834-6
ORIGINAL RESEARCH
Differential Gene Expression Associated withHoney Bee
Grooming Behavior inResponse toVarroa Mites
MollahMd.Hamiduzzaman1· BernaEmsen2· GregJ.Hunt3·
SubhashreeSubramanyam4· ChristieE.Williams4,5· JenniferM.Tsuruda6·
ErnestoGuzman‑Novoa1
Received: 15 August 2016 / Accepted: 3 January 2017 / Published online: 3 February 2017
© The Author(s) 2017. This article is published with open access at Springerlink.com
in NG, but it was not different from that of control bees.
The abundance of vitellogenin mRNA was not changed by
grooming activity. However, the abundance of blue cheese
mRNA was significantly reduced in IG compared to LG
or NG, but not to control bees. Efficient removal of mites
by IG correlated with different gene expression patterns
in bees. These results suggest that the level of grooming
behavior may be related to the expression pattern of vital
honey bee genes. Neurexin-1, in particular, might be useful
as a bio-marker for behavioral traits in bees.
Keywords Grooming behavior· Apis mellifera·
Gene expression· Varroa destructor· Neurexin, mRNA
abundance
Introduction
The parasitic mite Varroa destructor has caused the loss of
millions of honey bee (Apis mellifera) colonies and thus is
considered the number one health problem of honey bees
worldwide (Stankus 2008; Guzman-Novoa etal. 2010; Le
Conte et al. 2010). Varroa mites weaken bees by feeding
on their haemolymph after wounding their cuticle, which
may result in the invasion of secondary pathogens, lead-
ing to their early death (De Jong etal. 1982). Varroa mites
also suppress bee immunity (Yang and Cox-Foster 2005;
Navajas et al. 2008; Nazzi et al. 2012) and act as vectors
of several honey bee viruses (Kevan et al. 2006; Emsen
et al. 2015; Hamiduzzaman et al. 2015; Anguiano-Baez
etal. 2016). On the behavioral level, Varroa hampers non-
associative learning (Kralj et al. 2007), and reduces the
proportion of foragers that return to the hive (Kralj and
Fuchs 2006). Control of Varroa infestations in honey bee
colonies has become a daunting task for beekeepers and
Abstract Honey bee (Apis mellifera) grooming behavior
is an important mechanism of resistance against the para-
sitic mite Varroa destructor. This research was conducted
to study associations between grooming behavior and the
expression of selected immune, neural, detoxification,
developmental and health-related genes. Individual bees
tested in a laboratory assay for various levels of grooming
behavior in response to V. destructor were also analyzed for
gene expression. Intense groomers (IG) were most efficient
in that they needed significantly less time to start grooming
and fewer grooming attempts to successfully remove mites
from their bodies than did light groomers (LG). In addition,
the relative abundance of the neurexin-1 mRNA, was sig-
nificantly higher in IG than in LG, no groomers (NG) or
control (bees without mite). The abundance of poly U bind-
ing factor kd 68 and cytochrome p450 mRNAs were signifi-
cantly higher in IG than in control bees. The abundance of
hymenoptaecin mRNA was significantly higher in IG than
Edited by Yoon-Mi Hur.
* Ernesto Guzman-Novoa
eguzman@uoguelph.ca
1 School ofEnvironmental Sciences, University ofGuelph, 50
Stone Road East, Guelph, ONN1G2W1, Canada
2 Department ofAnimal Science, Ataturk University,
25240Erzurum, Turkey
3 Department ofEntomology, Purdue University, 901 West
State Street, WestLafayette, IN47907, USA
4 Department ofAgronomy, Purdue University, 915 West State
Street, WestLafayette, IN47907, USA
5 Crop Production andPest Control Research Unit, USDA-
ARS, WestLafayette, IN47907, USA
6 Clemson University, 130 McGinty Ct, Clemson, SC29634,
USA
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336 Behav Genet (2017) 47:335–344
1 3
scientists. Most beekeepers use synthetic miticides to con-
trol the parasites, but the continuous use of pesticides leads
to the development of resistance in the mites (Milani 1999).
Furthermore, the use of pesticides increases the risk of
contamination of honey and other hive products (Wallner
1999). Other ways of controlling this mite are thus needed.
One potential approach to controlling V. destructor would
be the development of honey bee strains resistant to the
parasite. This could theoretically be achieved by natural
selection (bees not treated against the mite) or by breed-
ing bees expressing traits associated to mite resistance or
tolerance (Rinderer etal. 2010; Arechavaleta-Velasco etal.
2012; Guzman-Novoa etal. 2012; Hunt etal. 2016).
The original host of V. destructor, the Asiatic bee Apis
cerana, naturally resists infestations by Varroa through
multiple mechanisms. The most important mechanism of A.
cerana resistance appears to be through grooming behav-
ior (Peng etal. 1987). The western honey bee, A. mellifera,
also expresses grooming behavior against Varroa, but to
a lesser degree than its Asiatic counterpart (Buchler et al.
1992; Fries etal. 1996). Through grooming behavior, some
adult bees physically remove mites from their bodies using
their legs and mandibles (Ruttner and Hanel 1992; Fries
etal. 1996; Boecking and Spivak 1999; Bahreini and Cur-
rie 2015). Grooming behavior is also a defense mechanism
against tracheal mites (Pettis and Pankiw 1998; Danka and
Villa 2003, 2005).
Bees groom themselves at various levels of intensity.
Guzman-Novoa etal. (2012) reported that bees that groom
at high intensity remove significantly more mites from their
bodies than bees that do it lightly, suggesting that grooming
intensity is an important factor for resistance to Varroa. Not
much is known about the genetic mechanisms regulating
grooming behavior but it appears to be a quantitative trait
with a genetic component (Moretto etal. 1993; Page and
Guzman-Novoa 1997; Arechavaleta-Velasco et al. 2012).
Grooming behavior is also influenced by environmental
effects (Currie and Tahmasbi 2008). The degree to which
grooming behavior is influenced by genes is unknown but,
if there is significant genetic variability for this trait, bees
could be bred for high grooming expression and intensity to
develop resistant stock to V. destructor (Hunt etal. 2016).
A number of studies have shown that V. destructor
parasitism alters the expression pattern of immune-related
(Yang and Cox-Foster 2005; Navajas etal. 2008; Hamiduz-
zaman et al. 2012) and behavioral-related genes in honey
bees (Le Conte et al. 2011). However, there are no stud-
ies of gene expression in bees that exhibit intense grooming
behavior. To learn more about genes that may be involved in
bee behavioral mechanisms of resistance against mites, we
explored the association of different degrees of grooming
behavior with mRNA abundance of some candidate genes
for which expression information exists for other traits, and
from some genes tested for the first time. We chose genes
that have reduced expression in response to V. destructor
parasitism such as the immune related gene, hymenoptae-
cin (Hym), the putative cell proliferation regulator, poly U
binding factor kd 68 (pUf68), and a gene related to longev-
ity, development and general health, vitellogenin (Vg). We
also tested a gene for the autophagy-linked FYVE protein,
blue cheese (BlCh), whose expression is changed by V.
destructor parasitism (Yang and Cox-Foster 2005; Navajas
etal. 2008; Dainat etal. 2012; Hamiduzzaman etal. 2012).
Honey bees like other insects rely on detoxification genes
such as the cytochrome p450 gene, CYP9Q3, which has
shown altered expression patterns when insects are exposed
to different types of chemicals (Mao etal. 2011), or when
performing physical activities such as hygienic behavior
(Boutin et al. 2015). But the expression of CYP9Q3 has
not been assessed for bees that are exposed to mites or as
a response to other behavioral activities such as groom-
ing behavior. Expression of the neural gene neurexin-1
(AmNrx1) occurs primarily in the central nervous system
and in the mushroom body of the brain, which is an impor-
tant organ for higher-order processing and learning in the
bee (Heisenberg 1998; Szyska etal. 2008) and AmNrx1 is
among a small number of candidate genes for honey bee
grooming behavior identified in a quantitative trait locus for
honey bee grooming behavior (Arechavaleta-Velasco etal.
2012). AmNrx1 is also known to be related to autism dis-
order in humans, a syndrome that is associated with repeti-
tive movements or ataxias (Feng etal. 2006; Sudhof 2008;
Reichelt etal. 2012) and in self-grooming behavior in mice
(Etherton etal. 2009). Therefore this gene could potentially
affect grooming behavior, but has not been studied in rela-
tion to this trait in bees.
The objectives of this study were (1) to correlate the
effect of two levels of grooming behavior (light and
intense) with the time required to start grooming and with
the number of attempts needed by individual bees exposed
to Varroa mites to successfully remove the parasite from
their bodies, and (2) to analyze the association between
these levels of grooming behavior and the expression of
selected genes in tested bees.
Materials andMethods
Collection ofV. destructor Mites
Grooming experiments were conducted at the Honey Bee
Research Centre of the University of Guelph, in Guelph,
Ontario, Canada between April and August, 2013. Adult
foundress Varroa mites from heavily infested honey bee
colonies were harvested from brood cells containing white-
eyed pupae using a fine paint brush. The harvested mites
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337Behav Genet (2017) 47:335–344
1 3
were held in Petri dishes lined with moist filter paper and
containing two white-eyed bee pupae collected from a non-
infested colony; the pupae served as a food source for the
mites. The mites were kept at room temperature (26 ± 2 °C)
and used within 2h from the time of collection.
Grooming Behavior inIndividual Bees
Grooming behavior at the individual level was performed
in the laboratory using a modified version of the method
described by Aumeier (2000). All worker bees were sam-
pled from five local, randomly selected colonies, presum-
ably representing a broad sample of genotypes because
queens of the colonies were open mated to approximately
12–20 haploid drones. Worker bees for all treatments
were collected from the brood nest of the source colonies
using a bee vacuum (Gary and Lorenzen 1990). Individual
Petri dishes (9 cm diameter) were prepared in advance
of the assays by lining their bottom with a circular piece
of white filter paper to provide contrast for observation
of bees and mites. Petri dishes were covered with plastic
wrap. The plastic wrap was perforated 20–30 times with a
nail (50 × 3mm) in order to allow air to pass through. One
worker bee was introduced into each dish and was then
given 2–3min to become accustomed to the Petri dish. The
plastic wrap was then lifted slightly in order to place a sin-
gle mite on the bee’s thorax using a fine brush (except for
control bees that were only touched with the brush on the
thorax). A stopwatch was started immediately upon appli-
cation of the mite and the bee was observed for up to 3min.
Grooming instances exhibited by the bee were recorded,
specifically describing the time elapsed until she started
to groom, the number of grooming attempts, whether or
not she removed the mite and the intensity with which she
groomed. The following variables were recorded: time (s)
elapsed from the moment a mite was placed on the bee tho-
rax until she started to groom, time to mite removal, and
the number of grooming attempts a bee required to suc-
cessfully remove a mite. A grooming attempt was defined
as an uninterrupted period of time during which groom-
ing was observed, and that ended when the bee paused (a
bee could have several of these grooming instances within
3 min). In the event that a bee successfully removed the
mite within 3min, the trial ended and the time of removal
was recorded. Bees that could not remove the mite within
3min were only classified by the intensity with which they
groomed. “Light grooming” (LG) consisted of slow swipes
of one or occasionally two legs across the thorax or abdo-
men. “Intense grooming” (IG) consisted of vigorous wip-
ing and shaking and always involved the use of more than
two legs. Whether the grooming was recorded as “light”
or “intense” and how many grooming attempts were per-
formed by each bee was left to the observer’s judgement.
However, there was only one observer, and therefore all
incidences were judged by the same person as described
by Guzman-Novoa etal. (2012). Some bees did not groom
and were recorded as “no grooming” (NG). Since control
bees were not exposed to the irritation caused by Varroa
mites and were not assessed for mite removal, they were
only evaluated for whether or not they groomed, and for
those that groomed, the time to start grooming and groom-
ing attempts within 3min were recorded. Grooming trials
were performed with a total of 240 bees. Samples of 12–16
individuals for each IG, LG, NG and control bees were ran-
domly collected at the end of trials and frozen at −70 °C for
further analysis of gene expression.
RNA Extraction andcDNA Synthesis
Total RNA was extracted by homogenizing each adult bee
sample in extraction buffer as per Chen etal. (2000). The
homogenates were extracted twice with chloroform and
the RNA was precipitated using LiCl as described by Sam-
brook et al. (1989). The amount of total RNA extracted
was determined with a spectrophotometer (Nanovue GE
Healthcare, Cambridge, UK). RNA samples were stored
at−70 °C. For cDNA synthesis, 2 µg of total RNA was
reverse-transcribed using Oligo (dT)18 and M-MuLV RT
with the RevertAid™ H Minus First Strand cDNA Synthe-
sis Kit (Fermentas Life Sciences, Burlington, ON, Canada),
following the instructions of the manufacturer. The cDNA
was stored at −20 °C.
Primers
The primers used to amplify the genes evaluated are shown
in Table1. To design some of the primers, the complete
sequences of the genes were obtained from the National
Centre for Biotechnology Information (NCBI) (http://
www.ncbi.nlm.nih.gov). The sequences were aligned using
CLUSTALX and the primers were designed using the Gene
Runner (Version 3.05, Hastings Software, Inc., NY). The
oligo nucleotides were ordered from Laboratory Services
of the University of Guelph (Guelph, ON, Canada).
PCR Amplifications
Each of the target genes (except CYP9Q3) was co-ampli-
fied together with the honey bee ribosomal protein RpS5
gene (Thompson etal. 2007) in the same tube and reaction
as a constitutive control. The glyceraldehyde 3-phosphate
dehydrogenase2 (GAPD2) gene (Thompson et al. 2007)
was used as another standard control to co-amplify with
CYP9Q3. All PCR reactions were done with a Mastercy-
cler (Eppendorf, Mississauga, ON, Canada). Each 15µL of
reaction contained 1.5µL of 10× PCR buffer (New England
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338 Behav Genet (2017) 47:335–344
1 3
BioLabs, Pickering, ON, Canada), 0.5µL 10mM of dNTPs
(Bio Basic Inc., Markham, ON, Canada), 1µL of 10 µM
for each primer of target and housekeeping genes (Labora-
tory Services, University of Guelph, Guelph, ON, Canada),
0.2µL 5U/µL of Taq polymerase (New England BioLabs,
Pickering, ON, Canada), 2 µL of the cDNA sample, and
6.8µL of dd H2O. To amplify AmNrx1, CYP9Q3, Hym and
Vg, the thermocycler was programmed to run at 94 °C for
3min, followed by 35 cycles of 30s at 94 °C, 60s at 58 °C
and 60 s at 72 °C, and a final extension step at 72 °C for
10min. To amplify pUf68 and BlCh, the annealing temper-
ature was 55 °C while the other conditions described above
remained the same.
Separation andSemi‑Quantification ofPCR Products
PCR products were separated on 1% TAE agarose gels
and stained with ethidium bromide. A 100bp DNA ladder
(Bio Basic Inc., Markham, ON, Canada) was included in
each gel. Images of the gels were captured using a digital
camera with a Benchtop UV Transilluminator (BioDoc-ItM
Imaging System, Upland, CA). The intensity of the ampli-
fied bands was measured in pixels using the Scion Image
Program (Scion Corporation, Frederick, MD) as per Dean
etal. (2002). The ratio of band intensity between the target
gene and the housekeeping gene was calculated to deter-
mine the relative expression units (REU) of each gene. To
determine whether quantification at 35 amplification cycles
was not affected by signal saturation of the band intensi-
ties, randomly selected samples with high, medium and low
REUs were also quantified in the same manner with fewer
amplification cycles, and the pattern of expression based
on the REU values were not significantly different when
25, 30 and 35 amplification cycles were used (F2,15 = 0.30,
p = 0.75). We analyzed results at 35 cycles because in most
cases the relationship between the number of cycles and
molecules is relatively linear at 35 cycles when semi-quan-
titative RT-PCR is used, which provides high amplification
efficiency.
Quantitative Real‑Time‑PCR Methods
To confirm the correlation between AmNrx1 mRNA abun-
dance and grooming behavior obtained with the semi-
quantification method (this gene was the gene that most
consistently correlated with grooming behavior), target-
specific qRT-PCR primers (Table 1) corresponding to the
Neurexin1A gene were designed using the Primer Express
3.0 software (ABI, Applied Biosystems, Foster City, CA).
The qRT-PCR was performed using the Light Cycler 480
II Real Time PCR System (Roche, Indianapolis, IN) using
the SYBR Green dye-based detection system. All reactions
were performed in a final volume of 10µL, consisting of
5 µL of SensiFAST SYBR no-ROX master mix (Bioline,
Table 1 Primers used for amplification of the target and constitutive control genes
F forward primer, R reverse primer
*Target
**Constitutive control genes
Gene name Primer sequence (5′–3′) Gene ID Band size References
Hym*F: CTC TTC TGT GCC GTT GCA TA
R: GCG TCT CCT GTC ATT CCA TT
GB17538 200bp Evans (2006)
pUf68*F: CAA GAC CTC CAA CTA GCA TG
R: CAA CAG GTG GTG GTG GTG
GB13651 201bp Hamiduzzaman etal. (2012)
BlCh*F: GTG CTT GGG TTA GGA TGT GTAC
R: GTT AAT CTT CTT CCG CTA CTG
GB10249 218bp Hamiduzzaman etal. (2012)
AmNrx1*F: ACG CCC ACC ACA GAG ATG AC
R: CAT TTG GAT CCT GGC AGA AG
FJ580046 259bp This study
CYP9Q3*F: GTT CCG GGA AAA TGA CTA C
R: ACT CTC GAC GCA CAT CCT G
XM_006562300 296bp Mao etal. (2011)
This study
Vg*F: CTG TCG ATG GAG AAG GGA ACT
R: CTT GCC TAC GAG TCT TGC TGT
NM_001011578 370bp This study
RpS5** F: AAT TAT TTG GTC GCT GGA ATTG
R: TAA CGT CCA GCA GAA TGT GGTA
GB11132 115bp Evans (2006)
GAPD2** F: GAT GCA CCC ATG TTT GTT TG
R: TTT GCA GAA GGT GCA TCA AC
GB14798 203bp Thompson etal. (2007)
AmNrxn1*
(qRT-PCR)
F: ACG CCC ACC ACA GAG ATG AC
R: CCG ATT ATT AAG GCA GCG TTCT
FJ580046 137bp This study
AmRPL8**
(qRT-PCR)
F: TGG ATG TTC AAC AGG GTT CATA
R: CCG ATT ATT AAG GCA GCG TTCT
122bp This study
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339Behav Genet (2017) 47:335–344
1 3
Taunton, MA), gene-specific primers at a final concentra-
tion of 0.2µM each, and 20ng of cDNA template. No-tem-
plate and no-reverse transcriptase samples were included in
each PCR plate as negative controls. Along with the target-
gene, the qRT-PCR plate also included AmRPL8 (60S ribo-
somal protein L8) as an internal reference housekeeping
gene to verify equal amounts of target cDNA in all samples.
All reactions were set up in triplicate for each of the biolog-
ical replicates. PCR conditions were as follows: 95 °C for
5min, 45 cycles of 95 °C for 10s, 60 °C for 20s, and 72 °C
for 30s. To determine the specificity of the reaction a melt
curve analysis was carried out following PCR, confirming
amplification of a single product. Quantification of gene
expression, displayed as Relative Expression Value (REV)
was calculated using the Relative Standard Curve Method
(User Bulletin 2: ABI PRISM 7700 Sequence Detection
System) as described in Subramanyam etal. (2006).
Statistical Analysis
Data on time to start grooming, number of grooming
attempts, time to successful mite removal and gene expres-
sion were subjected to analysis of variance (ANOVA),
excluding non-groomers and negative control values from
the analyses because they represented 0 values. A correla-
tion analysis was performed with AmNrx1expression data
from the semi-quantification method and from the qRT-
PCR to validate results. To obtain descriptive statistics and
perform ANOVAS, the package IBM-SPSS v. 23 (SPSS
Inc., Chicago, IL) was used. Significant differences among
means were separated with Fisher’s protected LSD or Tam-
hane’s T2 tests (α = 0.05).
Results
IG bees started to groom themselves significantly
faster than LG and control bees. LG bees also initi-
ated grooming activity significantly faster than control
bees (F3, 206 = 220.83, p < 0.0001), whereas NG did not
groom at all within the 3 min lapse of the trial (Fig. 1).
To achieve mite removal success, IG bees required signifi-
cantly less time and fewer grooming attempts than LG bees
(F2, 177 = 76.50, p < 0.0001 and F2, 207 = 50.65, p < 0.0001
for time and removal attempts, respectively), whereas NG
bees did not groom or remove mites (Fig.2a, b), indicating
that IG bees are more efficient at removing mites than other
bees.
The expression of AmNrx1 was significantly higher in
IG than in LG, NG and control bees. There were no sig-
nificant differences in the level of expression of this gene
among LG, NG and control bees, indicating that only
intense grooming was associated with a high expression
level of AmNrx1 (F3, 48 = 12.20, p < 0.0001, Fig.3a).
The expression of pUf68 increased significantly in both
IG and LG bees relative to NG and control bees with no
differences between IG and LG bees. However, the level
of gene expression in NG was higher than in control bees
(F3, 60 = 20.94, p < 0.0001, Fig.3b).
The expression of CYP9Q3 was significantly higher in
IG than in control bees, but not different from that of NG
and LG bees (F3, 60 = 5.04, p < 0.01, Fig. 3c). Conversely
to the above results, the expression of BlCh was signifi-
cantly higher in LG and NG than in IG bees, while there
were no significant differences in expression of BlCh gene
Fig. 1 Mean time to start grooming ± SE (s) within 3 min in indi-
vidual worker bees either not exposed to V. destructor (control bees,
only touched with a fine brush on the thorax), or exposed to a mite
(by placing a mite on their bodies). Exposed bees responded by not
grooming (excluded from the analysis due to 0 values), or by groom-
ing at light pace (LG) or at vigorous pace (IG). Different letters indi-
cate significant differences of means based on analysis of variance
and Tamhane’s T2 tests (p < 0.01; n = 240)
Fig. 2 Mite removal success of worker bees exposed to V. destructor
for 3min in the laboratory. a Mean time spent for mite removal ± SE
(s) and b mean number of attempts until successful mite removal for
individual bees exposed to V. destructor by placing a mite on their
bodies. Only bees that responded by grooming at light pace (LG) or
at vigorous pace (IG) were included in the analysis. Different letters
indicate significant differences of means based on analysis of vari-
ance and Tamhane’s T2 tests (p < 0.01; n = 210)
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340 Behav Genet (2017) 47:335–344
1 3
between control bees and bees of the rest of the treatments
(F3, 60 = 5.45, p < 0.05, Fig. 3d). Hym was significantly
upregulated in IG compared to NG bees, but there were
no significant differences in gene expression levels among
NG, LG and control bees (F3, 36 = 4.12, p < 0.05, Fig.3e).
Finally, expression of Vg was not associated to grooming
behavior or the presence of Varroa, since no differences
in expression for this gene were observed among all treat-
ments (F3, 60 = 0.125, p > 0.05, Fig.3f).
The results from qRT-PCR of AmNrx1 supported those
obtained with the semi-quantitative method. IG bees had
higher AmNrx1 mRNA abundance than did LG and NG
bees (F2, 22 = 3.768, p < 0.05). Additionally, expression
data from the semi-quantification method and from the
qRT-PCR for the same bees were significantly correlated
(r = 0.65, p < 0.001).
Discussion
Bees that performed instances of intense grooming were
significantly faster to start grooming and required fewer
grooming attempts and less time to remove Varroa mites
from their bodies than bees performing light grooming.
These results indicated that IG bees were very sensitive
to the mite presence on their bodies and were efficient at
removing them. Guzman-Novoa et al. (2012) compared
different presumably Varroa-susceptible and resistant
genotypes of honey bees for grooming ability, and found
that a significantly higher number of mites were dis-
lodged from the bees’ bodies by intense grooming than
by light grooming regardless of genotype, which agrees
with the findings here reported.
Grooming behavior allows insects to clean their body
surface and sensory organs (Zhukovskaya et al. 2013).
Therefore, this behavior is linked with the ability of the
insect to perceive stimuli from its environment. Parasitic
mites provide mechanical and chemosensory stimuli,
which may result in the initiation of grooming behavior
by the affected bee. Thus, sensory recognition of the par-
asite could lead to behavioral and immune responses such
as grooming behavior (Roode and Lefevre 2012). More
efficient grooming bees may rely on quick recognition
of Varroa presence by tactile or chemosensory sensors.
This in turn would activate defense mechanisms, includ-
ing reacting through physical activities such as groom-
ing behavior, to successfully remove the mites from their
bodies. The age and reproductive status of mites could
also be a factor that influences the sensitivity of honey
bees to perform grooming behavior. Kirrane etal. (2012)
evaluated in laboratory cages the grooming response
of honey bees to V. destructor, and concluded that the
grooming success of bees was affected by the age and
reproductive status of the mites. The highest mite drop
was for daughter mites and the lowest for foundress mites,
which suggests that the former age group stimulated bees
to remove mites from their bodies more frequently than
when parasitized with foundresses. We used foundress
mites in our study, so, perhaps had we used only daugh-
ter mites we would have seen a higher frequency of mite
removal and probably higher levels of gene expression.
This hypothesis however, remains to be tested.
Supporting the above potential explanations, Biswas
et al. (2010) reported that the expression of the neural
gene AmNrx1 was affected by sensory experience in honey
bees, which may play a role in the development of synaptic
connections that could influence learning and the expres-
sion of behavioral traits. Also, Arechavaleta-Velasco etal.
(2012) demonstrated that some candidate genes, includ-
ing AmNrx1, were associated with grooming behavior.
Similarly, successful mite removal by IG bees in this study
suggested that these bees may have a higher sensitivity to
Varroa, resulting in increased expression of neuron-related
genes, such as AmNrx1. The significantly higher expression
level of AmNrx1 in IG than in LG, NG and control bees
supported results from the above studies and the notion
that this gene is associated with grooming behavior and/
Fig. 3 Relative RT-PCR quantification units of AmNrx1 (a), pUf68
(b), CYP9Q3 (c), BlCh (d), Hym (e) and Vg (f), relative to house-
keeping genes (RpS5 or GAPD2) of individual worker bees not
exposed to V. destructor (control bees, only touched with a fine brush
on the thorax) or exposed to it (by placing a mite on their bodies).
Exposed bees responded by not grooming (NG), or by grooming at
light pace (LG) or at vigorous pace (IG). Different letters indicate
significant differences of means based on analysis of variance and
Fisher’s protected LSD tests (p < 0.05; n = 64 for all genes, except for
AmNrx1 with n = 52 and Hym with n = 40)
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341Behav Genet (2017) 47:335–344
1 3
or physical activity. Further study is needed to distinguish
between AmNrx1 effects on grooming or activity states.
The putative cell proliferation regulator protein, pUf68,
also known as half pint, plays important regulatory roles in
controlling the production of complex diverse proteins in
a wide range of organisms (Bourgeois etal. 2004). pUf68
is particularly known for its role in pre-mRNA splicing,
which could possibly be related to physical activity in the
insect. It might be that products of pUf68 are linked to
functions of the peripheral nervous system (PNS) of bees.
Physical activity such as grooming behavior in bees might
have an impact on the splicing of pUf68 and transcript
proliferation in cells through the PNS. The significantly
higher expression of pUf68 in both IG and LG than in
NG and control bees suggested that it could be affected by
grooming activity or vice versa. Contrary to our results, the
expression of pUf68 was found to be suppressed by Varroa
parasitism in adult bees (Yang and Cox Foster 2005; Nava-
jas etal. 2008) and brood (Dainat etal. 2012; Hamiduzza-
man etal. 2012). Perhaps the difference between our results
and those of the above studies is related to time of expo-
sure to the mite. In our study, bees were exposed to Varroa
<3min and so, presumably the mite did not have time to
inoculate immune-suppressive effectors through its saliva
while feeding on the bees’ haemolymph (Yang and Cox-
Foster 2007; Richards etal. 2011). Therefore, the mite may
have been unable to suppress the expression of this immune
related gene in the bees. Probably the high physical activ-
ity of grooming bees, leads to physiological changes result-
ing in higher expression of pUf68. It is also possible that
the expression of this gene unchains higher physical activ-
ity through neural mechanisms stimulated by the presence
of a mite. Regardless of why the expression of this gene
is affected, this is the first report of a relationship between
pUf68 mRNA abundance and grooming behavior in bees.
Further studies will be needed to clarify the mechanisms
through which grooming activity and the expression of this
gene in honey bees are related.
Expression of the detoxification gene, CYP9Q3, in IG
bees was significantly higher than in control bees, but simi-
lar to that of LG and NG bees. These results suggested an
effect on gene expression related to the presence of Varroa
on the bee’s body (since control bees were treated identi-
cally but not challenged with a mite) but not necessarily
associated with the physical activity of grooming behav-
ior. It may be that the short exposure to the mite unchains
a physiological reaction leading to a higher expression of
this gene only in bees exposed to the mite regardless of
their physical activity. Perhaps expression of CYP9Q3
can respond to a non-chemical stress, such as the attach-
ment of a Varroa mite (Mao et al. 2011; Boncristiani
et al. 2012). Supporting the hypothesis that CYP9Q3 is
not related to physical activity, Boutin etal. (2015) found
that cytochrome p450 genes were over-expressed in non-
hygienic bees compared to hygienic bees, and hypothesized
that the products of these genes degrade the odorant phero-
mones and chemicals that signal the presence of diseased
brood and thus resulted in these bees being less efficient in
detecting killed brood. Although no studies have been con-
ducted to demonstrate a relationship between mite odors
and grooming behavior, it is possible that the increased
expression of CYP9Q3 in our study had been influenced by
scents of the mite. Odorant substances such as pheromones
may influence gene expression in the honey bee. For exam-
ple, Grozinger etal. (2003) reported that queen mandibular
pheromone (QMP) affects gene expression in the bee brain,
which showed correlation with behavioral responses (i.e.
brood care, nursing) in adult worker bees.
Navajas etal. (2008) reported that the expression of the
autophagy-linked gene BlCh, was up-regulated in bees pre-
sumed to be Varroa-tolerant, while the expression of Dlic2
and Atg18 genes, which influence neural reactions, was
down-regulated. Interestingly, in another study, the expres-
sion of BlCh was negatively correlated with Dlic2 and
Atg18 in Varroa-parasitized bees (Simonsen et al. 2007).
These findings agree with our results of increased BlCh
expression in NG and LG bees and of decreased expres-
sion of this gene in IG bees. Intense physical activity dur-
ing grooming could be related to the nervous system being
stimulated by the products of Dlic2 and Atg18 genes, which
would also result in suppression of BlCh in IG bees. Future
experiments however, are required to confirm whether this
explanation is plausible.
The expression of Hym in IG bees was similar to that
of LG and control bees, but it was lower in NG bees. This
result is difficult to explain but perhaps it is related to dif-
ferences in activity between the groups of bees. Control
bees as well as LG and IG bees all groomed (and thus were
active), whereas NG bees showed reduced activity. It also
seems that mite parasitism had no effect on Hym expression
since control bees were not exposed to mites but did not
differ from LG and IG bees that were parasitized by a mite.
Another possibility is that mRNA abundance of genes such
as Hym, CYP9Q3 and AmNrx1 are all increased by stress,
which in turn increases the tendency for intense groom-
ing. Genotypic variation between bees of different sources
could also differentially influence gene expression in Var-
roa-parasitized and not parasitized bees (Navajas et al.
2008). However, these and other potential explanations of
our results, require further experimentation.
There was no significant difference in the expression
of the developmental and general health related gene, Vg,
among bees of the different treatments, indicating that nei-
ther physical activity nor short exposure to Varroa affects
the expression of this gene and that this gene does not seem
to be related to grooming behavior.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
342 Behav Genet (2017) 47:335–344
1 3
Because Varroa poses a serious threat to bee health,
researchers have been trying to find mite-resistance
traits in bees. Several studies have indicated that groom-
ing behavior may be a very important trait in conferring
resistance to bees against the mite at the colony and indi-
vidual levels (Moretto etal. 1993; Arechavaleta-Velasco
and Guzman-Novoa 2001; Andino and Hunt 2011; Hunt
etal. 2016; Invernizzi etal. 2016). These and a previous
study (Guzman-Novoa etal. 2012) demonstrate and con-
firm the importance of efficient grooming for successful
mite removal in honey bees. At the molecular level, Are-
chavaleta-Velasco etal. (2012) searched for genes influ-
encing grooming behavior by analyzing the DNA of bee
genotypes in backcross workers derived from high- and
low-grooming parents. These workers varied in tendency
to initiate grooming instances after being challenged with
Varroa mites on their bodies. These researchers identified
a single chromosomal region containing a set of candi-
date genes, which includes AmNrx1, using quantitative-
trait-locus (QTL) interval mapping. Consistent with this
finding, of all the genes tested in this study, AmNrx1 was
most highly and consistently related to intense grooming
and thus, warrants further investigation.
One limitation of this study is the small number of
genes selected to study in the context of grooming behav-
ior. Analyzing more genes based on their specific func-
tion might have been more informative in evaluating their
expression pattern during grooming instances. Despite
this limitation, some of the selected genes showed asso-
ciation with IG, indicating that probably multiple genes
rather than a single gene might be involved in regulat-
ing grooming behavior. However, whether the genes are
influencing the behavior or vice versa still needs to be
confirmed. Therefore, more studies need to be conducted
to understand the involvement of some of these and other
genes that are related to neural sensitivity as they respond
to the irritation caused by ectoparasitic mites on the bees.
Finding candidate genes that influence the intensity with
which bees groom themselves in response to parasitic
mites is critical for developing marker assisted selection
assays to breed for mite resistance in honey bees.
Acknowledgements We thank Paul Kelly for managing the colonies
and for supplying the brood and mites used in these experiments. This
study was partially funded by a Natural Sciences and Engineering
Research Council of Canada (NSERC) discovery grant to EG (Grant
400571) and by a grant from the United States Department of Agri-
culture (USDA) Grant 2008-35302-18803 to GJH.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no competing
interests.
Ethical approval All procedures performed in studies were in
accordance with the ethical standards.
Human and animal rights and Informed consent This article does
not contain any studies with human participants or animals performed
by any of the authors.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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