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Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites

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  • Food Industry, ON, Canada

Abstract and Figures

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic 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 significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than 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.
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Vol.:(0123456789)
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Behav Genet (2017) 47:335–344
DOI 10.1007/s10519-017-9834-6
ORIGINAL RESEARCH
Differential Gene Expression Associated withHoney Bee
Grooming Behavior inResponse toVarroa Mites
MollahMd.Hamiduzzaman1· BernaEmsen2· GregJ.Hunt3·
SubhashreeSubramanyam4· ChristieE.Williams4,5· JenniferM.Tsuruda6·
ErnestoGuzman‑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 etal. 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 etal. 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
etal. 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 ofEnvironmental Sciences, University ofGuelph, 50
Stone Road East, Guelph, ONN1G2W1, Canada
2 Department ofAnimal Science, Ataturk University,
25240Erzurum, Turkey
3 Department ofEntomology, Purdue University, 901 West
State Street, WestLafayette, IN47907, USA
4 Department ofAgronomy, Purdue University, 915 West State
Street, WestLafayette, IN47907, USA
5 Crop Production andPest Control Research Unit, USDA-
ARS, WestLafayette, IN47907, USA
6 Clemson University, 130 McGinty Ct, Clemson, SC29634,
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 etal. 2010; Arechavaleta-Velasco etal.
2012; Guzman-Novoa etal. 2012; Hunt etal. 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 etal. 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 etal. 1996). Through grooming behavior, some
adult bees physically remove mites from their bodies using
their legs and mandibles (Ruttner and Hanel 1992; Fries
etal. 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 etal. (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 etal. 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 etal. 2016).
A number of studies have shown that V. destructor
parasitism alters the expression pattern of immune-related
(Yang and Cox-Foster 2005; Navajas etal. 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
etal. 2008; Dainat etal. 2012; Hamiduzzaman etal. 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 etal. 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 etal. 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 etal.
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 etal. 2006; Sudhof 2008;
Reichelt etal. 2012) and in self-grooming behavior in mice
(Etherton etal. 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 andMethods
Collection ofV. 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 2h from the time of collection.
Grooming Behavior inIndividual 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 × 3mm) in order to allow air to pass through. One
worker bee was introduced into each dish and was then
given 2–3min 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 3min.
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 3min, the trial ended and the time of removal
was recorded. Bees that could not remove the mite within
3min 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 etal. (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 3min 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 andcDNA Synthesis
Total RNA was extracted by homogenizing each adult bee
sample in extraction buffer as per Chen etal. (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 Table1. 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 etal. 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 15µL of
reaction contained 1.5µL of 10× PCR buffer (New England
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338 Behav Genet (2017) 47:335–344
1 3
BioLabs, Pickering, ON, Canada), 0.5µL 10mM of dNTPs
(Bio Basic Inc., Markham, ON, Canada), 1µL of 10 µM
for each primer of target and housekeeping genes (Labora-
tory Services, University of Guelph, Guelph, ON, Canada),
0.2µL 5U/µL of Taq polymerase (New England BioLabs,
Pickering, ON, Canada), 2 µL of the cDNA sample, and
6.8µL of dd H2O. To amplify AmNrx1, CYP9Q3, Hym and
Vg, the thermocycler was programmed to run at 94 °C for
3min, followed by 35 cycles of 30s at 94 °C, 60s at 58 °C
and 60 s at 72 °C, and a final extension step at 72 °C for
10min. To amplify pUf68 and BlCh, the annealing temper-
ature was 55 °C while the other conditions described above
remained the same.
Separation andSemi‑Quantification ofPCR Products
PCR products were separated on 1% TAE agarose gels
and stained with ethidium bromide. A 100bp 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
etal. (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 10µL, 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 200bp Evans (2006)
pUf68*F: CAA GAC CTC CAA CTA GCA TG
R: CAA CAG GTG GTG GTG GTG
GB13651 201bp Hamiduzzaman etal. (2012)
BlCh*F: GTG CTT GGG TTA GGA TGT GTAC
R: GTT AAT CTT CTT CCG CTA CTG
GB10249 218bp Hamiduzzaman etal. (2012)
AmNrx1*F: ACG CCC ACC ACA GAG ATG AC
R: CAT TTG GAT CCT GGC AGA AG
FJ580046 259bp This study
CYP9Q3*F: GTT CCG GGA AAA TGA CTA C
R: ACT CTC GAC GCA CAT CCT G
XM_006562300 296bp Mao etal. (2011)
This study
Vg*F: CTG TCG ATG GAG AAG GGA ACT
R: CTT GCC TAC GAG TCT TGC TGT
NM_001011578 370bp This study
RpS5** F: AAT TAT TTG GTC GCT GGA ATTG
R: TAA CGT CCA GCA GAA TGT GGTA
GB11132 115bp Evans (2006)
GAPD2** F: GAT GCA CCC ATG TTT GTT TG
R: TTT GCA GAA GGT GCA TCA AC
GB14798 203bp Thompson etal. (2007)
AmNrxn1*
(qRT-PCR)
F: ACG CCC ACC ACA GAG ATG AC
R: CCG ATT ATT AAG GCA GCG TTCT
FJ580046 137bp This study
AmRPL8**
(qRT-PCR)
F: TGG ATG TTC AAC AGG GTT CATA
R: CCG ATT ATT AAG GCA GCG TTCT
122bp 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 0.2µM each, and 20ng 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
5min, 45 cycles of 95 °C for 10s, 60 °C for 20s, and 72 °C
for 30s. 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 etal. (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 3min 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 etal. (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 etal.
(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)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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 etal. 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 etal. 2008) and brood (Dainat etal. 2012; Hamiduzza-
man etal. 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
<3min 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 etal. 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 etal. (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 etal. (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 etal. (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 etal. 1993; Arechavaleta-Velasco
and Guzman-Novoa 2001; Andino and Hunt 2011; Hunt
etal. 2016; Invernizzi etal. 2016). These and a previous
study (Guzman-Novoa etal. 2012) demonstrate and con-
firm the importance of efficient grooming for successful
mite removal in honey bees. At the molecular level, Are-
chavaleta-Velasco etal. (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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... Quantitative real-time PCR qPCR was performed in a 20 µL reaction mixture consisting of 1X Sso Advanced TM SYBR Green supermix (Bio-Rad), 0.2 µL of each primer, and 1 µL (100 ng) of cDNA template (Fig. 5). The oligonucleotide primers for qPCR are shown in Table (1) (Hamiduzzaman et al., 2017). The reaction was carried out in 96-well plates using a Bio-Rad I cycler (Bio-Rad Crop., Hercules, CA.) programmed with the following temperature profile: 95 °C for 30 sec followed by 50 cycles of 95 °C for 5 sec, 60 °C for 30 sec, melt curve from 65 to 95 °C in 0.5 °C/5 sec increments. ...
... A different expression of the hygienic behaviour trait in the honeybee since we recorded different levels of brood removal in both honeybee experimental colonies. Our findings corroborate the results of a previous study of the pin-killed test , and others which found a different expression of hygienic behaviour between honey bee races, varroa infestation response (Boutin et al., 2015;Gempe et al., 2016;Hamiduzzaman et al., 2017 andNganso et al., 2017) None of the 18 study colonies had a mean removal level over 93.5 % after 48 h, which is a convenient threshold level above which colonies are considered fully hygienic. However, one colony had a mean of 93.5 % over the experiment. ...
... Moretto et al. (1993) using direct observation of grooming instances of mite-parasitized bees calculated a heritability index of 0.71 for this trait (Moretto et al., 1993). Moreover, candidate genes influencing grooming behavior have been pinpointed in AHB and EHB populations in different countries of the Americas (Arechavaleta-Velasco et al., Hamiduzzaman et al., 2017;Morfin et al., 2023a), which indicates genetic and heritable effects on the behavior. ...
... Conversely, a study conducted in Uruguay reported no differences in the proportion of dislodged mites between AHBs and EHBs, but AHBs performed more grooming instances than EHBs (Invernizzi et al., 2016). The rate of mite removal success in AHBs has been attributed to a more frequent and intense grooming compared to Varroa-susceptible or EHB genotypes (Aumeier, 2001;Guzman-Novoa et al., 2012;Hamiduzzaman et al., 2017;Morfin et al., 2020a). ...
Article
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The honey bee (Apis mellifera) parasitic mite, Varroa destructor, is considered one of the main causes of colony losses in European honey bee (EHB) populations around the world. However, some EHB and Africanized honey bee (AHB) populations (derived from the African subspecies A. m. scutellata) that inhabit tropical and subtropical regions of the Americas, have survived varroa mite infestations in the absence of acaricide treatments. It is conceivable to expect that these honey bee populations, which have been subjected to natural selection over decades, would have developed resistance against V. destructor or possess pre-existing adaptations that allow them to survive mite parasitism. Here, we present a comprehensive literature review describing the spread of V. destructor and the honey bee populations occurring in Latin America (LA), and summarize the evidence of resistance of those populations to V. destructor. We also analyze reports describing the potential mechanisms of mite resistance and how they operate in those honey bee populations. Studies of a few EHB, as well as of numerous AHB populations exhibiting resistance to V. destructor in LA, unveil the existence of evolutionary adaptations that restrain V. destructor population growth and provide insight into the current host-parasite relationship. This review supports the notion that selective breeding of local honey bee populations from LA could be a viable strategy to manage varroa mite infestations in colonies.
... Recently, Morfin et al. (2020) found that the expression of Nrx1 was significantly higher in honey bee colonies with high grooming behavior compared to control colonies and positively correlated with the proportion of damaged mites at the colony level. Similarly, Hamiduzzaman et al. (2017) detected a correlation between the expression level of Nrx1 and the intensity of grooming behavior, and proposed other candidate genes that may be associated with this trait, including the enzyme of the P450 complex cytochrome P450 9e2 (CYP9Q3) and the splicing factor 45 (Spf45). ...
... Varroa-resistant bees (exhibiting intense grooming behavior) may have a higher sensitivity to the mite (e.g., through rapid sensory recognition), leading to an increased expression of genes involved in the formation and maintenance of synapses, such as Nrx1. Our results are in line with those previously obtained by Hamiduzzaman et al. (2017). These authors analyzed colonies exhibiting high grooming behavior and observed higher expression levels of Nrx1 in bees with intense grooming behavior compared to bees with light grooming behavior against V. destructor. ...
Article
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Grooming behavior is one of the mechanisms of social immunity in Apis mellifera. This behavior has been proposed as an active strategy of honey bees to restrain the population growth of the ectoparasitic mite Varroa destructor in honey bee colonies. The characterization of honey bee stocks with high grooming behavior is of utmost importance for honey bee breeding programs to set the background for mite resistance biomarker-based selection. In this study, we analyzed the expression level of 11 candidate genes putatively involved in grooming and hygiene behaviors in adult workers from mite-resistant (R) and mite-susceptible (S) honey bee stocks. Heads and bodies of worker bees from both stocks, previously tested for grooming response to two treatments (mite infestation and a paintbrush touch control stimulus) were assessed by qPCR. In the head, R bees exposed to mite infestation showed higher levels of Nrx1 and Dop2 and lower levels of Obp3 than S bees. At the body level, R and S bees differed in the expression levels of Nrx1, Oa1, Obp4, Obp14, Obp16, Obp18, Spf45, CYP9Q3 , with no stimulus-specific pattern. Overall, our results suggest the involvement of some of the analyzed genes in the specific response to mite infestation, possibly related to the sensitivity and specificity of the R bee to this stimulus at the head level, while other genes would be involved in the non-specific motor response to irritants at the body level. The present study provides new insights into the characterization of the grooming behavior in a selected honey bee stock and increases the available information on its underlying molecular mechanisms. We discuss the putative functions and use of the assessed genes as potential tools for biomarker-assisted selection and improvement of Varroa mite control strategies in honey bee colonies.
... This region contained 27 genes, including potential neurodevelopmental and behavioral candidates such as neurexin-1, ataxin-3, and atlastin. Hamiduzzaman et al. (2017), invastegated the relationships between grooming behavior and the expressions of genes related to immunity, nervous system, and detoxification. In bees exhibiting intense grooming behavior, significant upregulation of Neurexin-1 expression, which plays a key role in synaptic information transmission and maintenance (Craig and Kang, 2007;Dean and Dresbach, 2006), has been identified. ...
Conference Paper
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Honey bees, known as eusocial organisms, have developed behavioral strategies for various purposes in the course of evolution. These strategies have been shaped through adaptation processes to fulfill important functions such as maintaining colony life, protecting their offspring, finding resources, and coping with various environmental challenges. Bee behaviors, particularly, encompass specific behavioral resistance mechanisms against diseases and ectoparasites. Behavioral resistance is generally examined under two main categories: hygienic and grooming behaviors. Hygienic behavior involves the detection of diseased or parasitized larvae and pupae, the removal of infected/infested brood, thus reducing the spread of infection/infestation. Grooming behavior is classified into two categories based on the performer: auto- grooming and allo- grooming. A uto- grooming refers to self - grooming behavior, while allo- grooming describes mutual grooming between two bees or the grooming of one bee by several bees acting socially together. This review emphasizes the potential of honey bee populations in struggling the honey bee ectoparasite Varroa destructor by examining how hygienic and grooming behaviors are influenced by genetic and environmental factors. Additionally, it can serve as an important resource to guide future research and to better understand the effects of Varroa destructor challenges on the sustainability of bee colony populations.
... Honeybees have developed behavioral mechanisms against parasitic mites like V. destructor, including grooming and uncapping & removing hygienic behaviors (Hamiduzzaman et al., 2017). Hygienic behaviors are known as a behavioral response of honeybee workers to spreading infections in the colony. ...
Article
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This study evaluated the grooming and uncapping & removing hygienic behaviors of the honeybee colonies of the West Azerbaijan province of Iran from April 2021 to October 2022. Eighty colonies of Iranian honeybees infected with Varroa mite from Mahabad, Urmia, Oshnavieh, and Khoy cities of West Azerbaijan province were selected and studied regarding grooming and uncapping & removing hygienic behaviors. The results showed that there is no significant difference between the studied cities in terms of grooming behavior. The results showed that the season affects the grooming behavior of honeybee colonies in the studied cities. Therefore, the grooming behavior of the studied honeybee population in summer was significantly higher than that of colonies in spring (P < 0.05). Comparing the means of uncapping & removing hygienic behaviors after 48 hours showed that the honeybee colonies of the studied cities significantly differ in terms of these behaviors (P < 0.05). So, the highest and lowest averages of uncapping & removing hygienic behaviors after 48 hours were observed in the honeybee colonies of Khoy and Oshnavieh cities, respectively. The results showed a positive correlation between hygienic behaviors and all the functional-behavioral characteristics of honeybee colonies in this research. Our finding showed that the Iranian honeybee colonies of West Azerbaijan province of Iran can defend themselves against the Varroa mite by performing both grooming and uncapping & removing hygienic behaviors. Therefore, it is possible to improve the level of these behaviors in the honeybee colonies of this province by implementing breeding programs.
... Their results showed that bees from the resistant genotypes performed significantly more instances of intense grooming, and a significantly higher number of mites were dislodged from the bees' bodies through intense grooming than through light grooming in all genotypes, suggesting that grooming intensity is an essential factor in resistance to Varroa mite [15]. The neural gene AmNrx1 (neurexin-1), identified in a quantitative trait locus, exhibits significantly higher expression in honeybees that display intense grooming, potentially making it a promising tool for marker-assisted selection of grooming behavior in the future [16,17]. ...
Article
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The grooming behavior of honeybees serves as a crucial auto-protective mechanism against Varroa mite infestations. Compared to Apis mellifera, Apis cerana demonstrates more effective grooming behavior in removing Varroa mites from the bodies of infested bees. However, the underlying mechanisms regulating grooming behavior remain elusive. In this study, we evaluated the efficacy of the auto-grooming behavior between A. cerana and A. mellifera and employed RNA-sequencing technology to identify differentially expressed genes (DEGs) in bee brains with varying degrees of grooming behavior intensity. We observed that A. cerana exhibited a higher frequency of mite removal between day 5 and day 15 compared to A. mellifera, with day-9 bees showing the highest frequency of mite removal in A. cerana. RNA-sequencing results revealed the differential expression of the HTR2A and SLC17A8 genes in A. cerana and the CCKAR and TpnC47D genes in A. mellifera. Subsequent homology analysis identified the HTR2A gene and SLC17A8 gene of A. cerana as homologous to the HTR2A gene and SLC17A7 gene of A. mellifera. These DEGs are annotated in the neuroactive ligand–receptor interaction pathway, the glutamatergic synaptic pathway, and the calcium signaling pathway. Moreover, CCKAR, TpnC47D, HTR2A, and SLC17A7 may be closely related to the auto-grooming behavior of A. mellifera, conferring resistance against Varroa infestation. Our results further explain the relationship between honeybee grooming behavior and brain function at the molecular level and provide a reference basis for further studies of the mechanism of honeybee grooming behavior.
... Yet, there were variations in the level of damaged mites across experimental colonies, and this would preclude us from concluding that grooming behavior is an effective mechanism of defense against mites. Because, several driving factors can considerably influence the degree of grooming behaviors among honeybee stocks, even for colonies existing in the same geographic region (Boecking et al., 2000;Masaquiza et al., 2021;Hamiduzzaman et al., 2017). Therefore, considering driving factors would be important during selection breeding programs to enhance resistant bee stocks. ...
Article
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The parasitic mite V. destructor has caused long-lasting losses to the survival of European honeybee colonies. In contrast, African honeybees are likely capable of surviving the effects of this parasitic mite with varying defense mechanisms. This study provides insights into two defense behavioral traits, including hygienic and grooming behaviors of local honeybee, Apis mellifera bandasii colonies against V. destructor mite in Ethiopia. Hygienic behavior (HB) was evaluated using the standard pin-killed brood method by calculating the dead brood removal rates (%) at 24 and 48 hrs. While grooming behavior (GB) was assessed by measuring the number of daily fallen mites and the percentage of damaged mites. The results of hygienic behavior showed greater brood removal rates of 83.1±14.3% and 97.6±3.4% at 24 hrs and 48 hrs, respectively. There were strong negative correlations between the HB and Varroa infestation rates, indicating that HB has the potential to reduce the mite population in colonies. Grooming behavior also showed higher mean daily fallen mites per colony (16.3±10.2), of which about 80% of the total fallen mites (n=488) were damaged. Ten body damage categories were identified, with most damages inflicted on mites' legs, dorsal shield, and gnathosoma because of the GB. Our study suggests that combined hygienic and grooming behaviors could be used as effective defenses against V. destructor infestations in A. m. bandasii colonies. Therefore, future selective breeding programs should integrate these specific host defenses in order to produce sustainable colonies resistant to this parasitic mite.
Article
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The Varroa destructor represents a significant threat to honey bees, leading to substantial yield losses and declines in colony health. Defense behaviors, including grooming (auto and allogrooming), serve as crucial mechanisms against Varroa infestations, yet the genetic basis of these behaviors remains elusive. This study examines the expression levels of hymenoptaecin (hym), neurexin-1 (AmNrx1), and CYP9Q3, potentially associated with defense behavior, in colonies of the Muğla honey bee ecotype (Apis mellifera anatoliaca) subjected to a Varroa selection program. Worker bees from 23 control groups and 23 colonies under selection were evaluated by using qPCR analysis. Results reveal a significant upregulation of hym, AmNrx1, and CYP9Q3 genes in the selected group, with fold changes of 2.9, 2.95, and 3.26 respectively compared to controls (p < 0.01). This suggests that selection against Varroa induces alterations in gene expression linked to Varroa exposure behaviors. These findings advocate for the potential use of hym, AmNrx1, and CYP9Q3 genes in preselection for future Varroa-resistant programs in honey bees. Supported by previous studies, these genes may facilitate the establishment of populations with enhanced defense behaviors, such as autogrooming and allogrooming.
Article
Honey bees use grooming to defend against the devastating parasite Varroa destructor Anderson and Trueman. We observed the grooming responses of individual bees from colonies previously chosen for high- and low-grooming behavior using a combination of mite mortality and mite damage. Our aim was to gain insight into specific aspects of grooming behavior to compare if high-grooming bees could discriminate between a standardized stimulus (chalk dust) and a stimulus of live Varroa mites and if bees from high-grooming colonies had greater sensitivity across different body regions than bees from low-grooming colonies. We hypothesized that individuals from high-grooming colonies would be more sensitive to both stimuli than bees from low-grooming colonies across different body regions and that bees would have a greater response to Varroa than a standardized irritant (chalk dust). Individuals from high-grooming colonies responded with longer bouts of intense grooming when either stimulus was applied to the head or thorax, compared to sham-stimulated controls, while bees from low-grooming colonies showed no differences between stimulated and sham-stimulated bees. Further, high-grooming bees from colonies with high mite damage exhibited greater grooming to Varroa than high-grooming colonies with only moderate mite damage rates. This study provides new insights into Varroa-specific aspects of grooming, showing that although a standardized stimulus (chalk dust) may be used to assess general grooming ability in individual bee grooming assays, it does not capture the same range of responses as a stimulus of Varroa. Thus, continuing to use Varroa mites in grooming assays should help select colonies with more precise sensitivity to Varroa.
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The Varroa destructor poses a significant threat to honey bees, leading to substantial yield losses and colony declines. Defence behaviour (such as grooming behavior: auto and allogrooming) in honey bees serves as a crucial mechanism against Varroa infestations, but the many genes responsible for this behavior remain unidentified. This study focuses on the expression levels of hymenoptaecin (Hym), neurexin-1 (AmNrx1) , and CYP9Q3 which could be associated with defence behavior, in Muğla honey bee ecotype ( Apis mellifera anatoliaca ) colonies subjected to a against Varroa selection program. Using the qPCR method, researchers analyzed worker bees from 23 control groups and 23 colonies under the selection program. The results revealed a remarkable increase in the expression levels of Hym, AmNrx1 , and CYP9Q3 genes in the selected group, with respective fold changes of 2.9, 2.95, and 3.26 compared to the control group (p < 0.01). This finding suggests that selection against Varroa infestations induces alterations in gene expression linked to behaviour related to exposure of Varroa in honey bees. These outcomes propose the potential use of Hym, AmNrx1 , and CYP9Q3 genes in preselection for future Varroa-resistant programs in honey bees. The genes used in the study that may be related to this behavior are supported by other studies in the future, they may help create an initial population with advanced defence behaviours (such as autogrooming and allogrooming).
Article
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The prevalence and loads of deformed wing virus (DWV) between honey bee (Apis mellifera L.) colonies from a tropical and a temperate environment were compared. The interaction between these environments and the mite Varroa destructor in relation to DWV prevalence, levels, and overt infections, was also analyzed. V. destructor rates were determined, and samples of mites, adult bees, brood parasitized with varroa mites and brood not infested by mites were analyzed. DWV was detected in 100% of the mites and its prevalence and loads in honey bees were significantly higher in colonies from the temperate climate than in colonies from the tropical climate. Significant interactions were found between climate and type of sample, with the highest levels of DWV found in varroa-parasitized brood from temperate climate colonies. Additionally, overt infections were observed only in the temperate climate. Varroa parasitism and DWV loads in bees from colonies with overt infections were significantly higher than in bees from colonies with covert infections. These results suggest that interactions between climate, V. destructor, and possibly other factors, may play a significant role in the prevalence and levels of DWV in honey bee colonies, as well as in the development of overt infections. Several hypotheses are discussed to explain these results.
Article
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The type and degree of damage to adult workers of Apis mellifera from infestation with the parasitic mite Varroa jacobsoni during development was investigated. Mean weights of infested bees upon emergence as adults were 6·3% to 25% less than for healthy bees. Mean % weight loss was correlated at a high level of significance with the number of mites in the cell. Only 6% of infested bees showed obvious physical deformation in the form of wing damage.
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Hygienic behavior is a complex, genetically-based quantitative trait that serves as a key defense mechanism against parasites and diseases in Apis mellifera. Yet, the genomic basis and functional pathways involved in the initiation of this behavior are still unclear. Deciphering the genomic basis of hygienic behavior is a prerequisite to developing an extensive repertoire of genetic markers associated to the performance level of this quantitative trait. To fill this knowledge gap, we performed an RNA-seq on brain samples of 25 honeybees per hives from five hygienic and three non-hygienic hives. This analysis revealed that a limited number of functional genes are involved in honeybee hygienic behavior. The genes identified, and especially their location in the honeybee genome, are consistent with previous findings. Indeed, the genomic sequences of most differentially expressed genes were found on the majority of the QTL regions associated to the hygienic behavior described in previous studies. According to the Gene Ontology annotation, 15 genes are linked to the GO-terms DNA or nucleotide binding, indicating a possible role of these genes in transcription regulation. Furthermore, GO-category enrichment analysis revealed that electron carrier activity is over-represented, involving only genes belonging to the cytochrome P450. Cytochrome P450 enzymes' overexpression can be explained by a disturbance in the regulation of expression induced by changes in transcription regulation or sensitivity to xenobiotics. Over-expressed cytochrome P450 enzymes could potentially degrade the odorant pheromones or chemicals that normally signal the presence of a diseased brood before activation of the removal process thereby inhibit hygienic behavior. These findings improve our understanding on the genetics basis of the hygienic behavior. Our results show that hygienic behavior relies on a limited set of genes linked to different regulation patterns (expression level and biological processes) associated with an over-expression of cytochrome P450 genes.
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Tliisstudy was uiidetlakcn to dctcniiiiic whcllícr Italiaii aiid AfricaJiizcdnieilifera workcre can ridÜicitísclvcs ofÜic initc Varroa Jacobsoni aftcr artificial infcstation with adull fciiialcs oftlús parasite.
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A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.
Article
The ectoparasitic mite Varroa destructor is one of the main plagues of honey bees Apis mellifera. Grooming behavior is a resistance mechanism through which parasitized bees can dislodge mites by themselves (autogrooming) or by the action of other bees (allogrooming). The objective of this study was to evaluate grooming behavior in Italian (A. m. ligustica) and Africanized (hybrids of A. m. scutellata) bees at the individual, group, and colony levels. Firstly, five behaviors were recorded observing bees individually placed on a Petri dish and after placing a mite on their thorax. Secondly, 30 bees of each colony were placed in a Petri dish along with 20 mites and 24 h later fallen mites were counted. Lastly, the proportion of injured mites collected in the hive floor was determined. At the individual level, Africanized bees showed a higher total number of reaction behaviors to V. destructor than did Italian bees (U = 182.5; p = 0.02). Groups of Italian bees could dislodge 60.8 ± 20.0% of mites and Africanized bees dislodged 65.9 ± 15.6% of mites, without showing significant differences (t = 0.735; p = 0.47). Colonies of Africanized bees showed a higher proportion of injured mites (29.0 ± 8.6%) than colonies of Italian bees did (17.7 ± 9.8%) (t = 2.92; p = 0.009). Africanized bees are characterized by presenting higher resistance to V. destructor than European bees. This study shows that such difference can be, partly due to grooming behavior. The importance of auto and allogrooming regarding resistance to V. destructor is discussed.
Article
The objective of this study was to assess the effects of honey bees (Apis mellifera L.) with different grooming ability and queen pheromone status on mortality rates of Varroa mites (Varroa destructor Anderson and Trueman), mite damage, and mortality rates of honey bees. Twenty-four small queenless colonies containing either stock selected for high rates of mite removal (n = 12) or unselected stock (n = 12) were maintained under constant darkness at 5 °C. Colonies were randomly assigned to be treated with one of three queen pheromone status treatments: (1) caged, mated queen, (2) a synthetic queen mandibular pheromone lure (QMP), or (3) queenless with no queen substitute. The results showed overall mite mortality rate was greater in stock selected for grooming than in unselected stock. There was a short term transitory increase in bee mortality rates in selected stock when compared to unselected stock. The presence of queen pheromone from either caged, mated queens or QMP enhanced mite removal from clusters of bees relative to queenless colonies over short periods of time and increased the variation in mite mortality over time relative to colonies without queen pheromone, but did not affect the proportion of damaged mites. The effects of source of bees on mite damage varied with time but damage to mites was not reliably related to mite mortality. In conclusion, this study showed differential mite removal of different stocks was possible under low temperature. Queen status should be considered when designing experiments using bioassays for grooming response.
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Devices and methods are described for capturing and sorting subpopulations of tagged adult honey bees, Apis mellifera L., that are commingled with large populations of untagged bees in experimental colonies. Each comb with adhering bees is placed on a rotatable frame-support device to facilitate visual examination. Bees are captured individually on the tip of a vacuum probe and released at one of many air-intake portholes that transport them safely to respective holding cages within a vacuum chamber. The escape of confined bees is prevented during the operation by a vacuum-actuated hinged door covering each porthole. These procedures facilitate handling, protect bees from mechanical injury, and minimize the risk of being stung.
Article
For the first time, adults and brood of Africanized and European honey bees (Apis mellifera) were compared for relative virus levels over 48 h following Varroa destructor parasitism or injection of V. destructor homogenate. Rates of increase of deformed wing virus (DWV) for Africanized versus European bees were temporarily lowered for 12 h with parasitism and sustainably lowered over the entire experiment (48 h) with homogenate injection in adults. The rates were also temporarily lowered for 24 h with parasitism but were not affected by homogenate injection in brood. Rates of increase of black queen cell virus (BQCV) for Africanized versus European bees were similar with parasitism but sustainably lowered over the entire experiment with homogenate injection in adults and were similar for parasitism and homogenate injection in brood. Analyses of sac brood bee virus and Israeli acute paralysis virus were limited as detection did not occur after both homogenate injection and parasitism treatment, or levels were not significantly higher than those following control buffer injection. Lower rates of replication of DWV and BQCV in Africanized bees shows that they may have greater viral resistance, at least early after treatment. Copyright © 2014. Published by Elsevier Inc.