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The ectoparasitic mite, Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera) mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. Several honeybee populations across Europe have well-documented adaptations of mite-resistant traits but little is known about host adaptations towards the virus infections vectored by the mite. The aim of this study was to assess and compare the possible contribution of adapted virus tolerance and/or resistance to the enhanced survival of four well-documented mite-resistant honeybee populations from Norway, Sweden, The Netherlands and France, in relation to unselected mite-susceptible honeybees. Caged adult bees and laboratory reared larvae, from colonies of these four populations, were inoculated with DWV and ABPV in a series of feeding infection experiments, while control groups received virus-free food. Virus infections were monitored using RT-qPCR assays in individuals sampled over a time course. In both adults and larvae the DWV and ABPV infection dynamics were nearly identical in all groups, but all mite-resistant honeybee populations had significantly higher survival rates compared to the mite-susceptible honeybees. These results suggest that adapted virus tolerance is an important component of survival mechanisms.
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Adapted tolerance to virus
infections in four geographically
distinct Varroa destructor‑resistant
honeybee populations
Barbara Locke1*, Srinivas Thaduri1, Jörg G. Stephan1, Matthew Low1, Tjeerd Blacquière2,
Bjørn Dahle3, Yves Le Conte4, Peter Neumann5,6 & Joachim R. de Miranda1
The ectoparasitic mite, Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera)
mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed
wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. Several honeybee populations
across Europe have well‑documented adaptations of mite‑resistant traits but little is known about
host adaptations towards the virus infections vectored by the mite. The aim of this study was to assess
and compare the possible contribution of adapted virus tolerance and/or resistance to the enhanced
survival of four well‑documented mite‑resistant honeybee populations from Norway, Sweden, The
Netherlands and France, in relation to unselected mite‑susceptible honeybees. Caged adult bees and
laboratory reared larvae, from colonies of these four populations, were inoculated with DWV and
ABPV in a series of feeding infection experiments, while control groups received virus‑free food. Virus
infections were monitored using RT‑qPCR assays in individuals sampled over a time course. In both
adults and larvae the DWV and ABPV infection dynamics were nearly identical in all groups, but all
mite‑resistant honeybee populations had signicantly higher survival rates compared to the mite‑
susceptible honeybees. These results suggest that adapted virus tolerance is an important component
of survival mechanisms.
e ectoparasitic mite, Varroa destructor, is inarguably the leading cause of honeybee (Apis mellifera) mortality
world-wide, practically exterminating wild colonies and severely aecting the management and protability of
beekeeping in the wake of its global spread during the 1980’s and 1990’s1. e damage this parasite causes to its
new host by feeding on adults and brood is amplied by the multiple viruses it carries and transmits24. Two
virus-complexes in particular are transmitted highly eciently by varroa mites with devastating consequences5:
the Deformed wing virus (DWV) complex, including major strains DWV-A, DWV-B and DWV-C6; and the Acute
bee paralysis virus (ABPV) complex, including major strains ABPV, Kashmir bee virus (KBV) and Israeli acute
paralysis virus (IAPV)7. Both DWV and ABPV are single-stranded RNA viruses that infect all stages of honeybee
development7, 8. In the absence of varroa mites, they are maintained in the colony at low levels as innocuous
infections through horizontal and vertical transmission routes914. DWV is the most common and wide spread
virus2. Symptoms are almost exclusively associated with varroa-mediated transmission when the mite feeds on
the bee during the pupal developmental stages8, 15 causing severe wing deformities that result in ightless adult
bees that die shortly aer emerging8. ABPV symptoms are characterized mostly by severe pupal mortality and
by trembling, paralysis and behavioural inadequacies of adult bees at elevated titres7.
A natural mite population in an infested honeybee colony can grow exponentially, rapidly leading to a DWV
and/or ABPV epidemic that ultimately precipitates the death of the colony typically within a few years5, 12, 16.
To avoid virus epidemics and thus colony death, mite population control strategies are essential in apiculture
in almost all parts of the world where the mite exists16. However, there are several extraordinary honeybee
populations in Europe and North America that have been documented to survive for extended periods without
OPEN
1Department of Ecology, Swedish Species Information Centre, Swedish University of Agricultural Sciences,
Uppsala, Sweden. 2Bio-Interaction and Plant Health, Wageningen University and Research, Wageningen,
The Netherlands. 3Department of Animal and Aquacultural Sciences, Norwegian University of Sciences,
Kløfta, Ås, Norway. 4Abeilles et Environnement, French National Institute for Agricultural Research, Avignon,
France. 5Vetsuisse Faculty, University of Bern, Bern, Switzerland. 6Agroscope, Swiss Bee Research Center, Bern,
Switzerland. *email: barbara.locke@slu.se
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mite control measures and without the harmful eects typically associated with varroa mite infestation17. ese
populations have all been exposed to the selection pressure of long-term uncontrolled mite infestation and in
response have adapted a repertoire of mite-resistant traits that limit the mite population growth rate1821. ese
traits subsequently support colony survival likely by reducing the transmission potential for lethal virus epidem-
ics. However, despite having acquired mite-resistant traits, these populations can still experience and survive
with occasional high mite infestation levels. Suggesting that perhaps other adapted survival mechanisms are
contributing to the long-term survival success of these populations such as host resistance or tolerance to the
actual mortality inducing virus infections.
In host–parasite interactions, host tolerance is dened as the ability to reduce the eect of the parasite, while
host resistance is the ability to reduce the tness of the parasite22. Recently, we have shown that individuals from
the naturally adapted mite-resistant honeybee population on Gotland, Sweden, survive with higher thresh-
olds of DWV and ABPV infections before bee health is compromised, relative to mite-susceptible unselected
honeybees23. is suggests that host tolerance, rather than resistance, to virus infections is an important compo-
nent of the naturally adapted survival mechanisms of the Gotland mite-resistant population, in addition to their
adapted mite-resistant traits. At the colony level, the Gotland honeybee population also appear to have adapted
resistance to other virus infection not directly transmitted by varroa mites but nevertheless harm honeybee
health and reduce long-term survival24, 25.
e aim of this study was to assess the possible contribution of adapted virus tolerance and/or resistance to
the enhanced survival of three other well-documented mite-resistant honeybee populations from Norway20,
e Netherlands21 and France17, 19, 26, while comparing them with the Gotland population in Sweden17, 18, 23 and
a non-selected mite-susceptible local honeybee population. is was done by comparing how experimental
APBV and DWV oral infections dierentially aect the larvae and adult bees from these honeybee population,
through both a virus infection time-course and adult bee mortality rates. Virus susceptibility was determined by
comparing the virus titres of virus-inoculated bees relative to both the pre-experiment background virus titres
and the natural infection development in uninoculated bees across the time-course. Adult bee mortality over
time was also recorded, as well as the virus titres in dead bees.
Materials and methods
Origin of honeybee colonies. e origin, management and varroa-resistance characteristics of the four
varroa-resistant populations has been abundantly described1821, 26, 27, and recently summarised in a review17.
Briey, the populations have evolved independently without mite control since 1994 (Avignon, France)26, 1999
(Gotland, Sweden)27, 2001 (Oslo, Norway)20 and 2005 (Tiengemeten, e Netherlands)21. During summer 2016,
twelve queens from each of the four mite-resistant populations were produced and mated in their geographic
locations of origin and were transported by surface courier to Sweden according to EU-legislation guidelines for
animal transport. Twelve queens from a local mite-susceptible population were similarly produced and mated,
to be used in this study as a control group. ese queens originated from an unselected population near Uppsala
that requires regular varroa mite control interventions by beekeepers to avoid colony death. Sixty host colonies,
each in a single full-size hive body containing 4 frames brood, 1 frame pollen, 2 frames honey and 3 frames wax
foundation, were acquired from four local beekeepers (15 colonies from each beekeeper) and placed in four
apiaries (one apiary per beekeeper) at the Lövsta Research Station at the Swedish University of Agricultural Sci-
ences in Uppsala, Sweden. e four apiaries were between 500 and 1000m separated from each other. On July
12, 2016, in each of the four apiaries, three queens from each of the ve populations (Control, Dutch, French,
Norwegian and Swedish) were introduced in the een colonies. All colonies were fed with a commercial 66%
w/w sugar solution with a 2/1/1 ratio of sucrose/fructose/glucose (Bifor®, Nordic Sugar A/S, Copenhagen, Den-
mark) to encourage queen acceptance. On August 25, 2016 each colony was treated against varroa with two
strips of tau-uvalinate (Apistan®, Vita Europe, UK) for six weeks, following the manufacturers recommended
procedures. No further varroa treatment was applied until the experiments described here began the following
summer in 2017, by which time all individuals in each colony were the ospring of the introduced experimen-
tal and control queens. e colonies used in these experiments were selected randomly from all four apiaries.
Samples of ~ 300 adult bees were taken from each colony at bi-monthly intervals during 2017 to determine the
phoretic varroa infestation rates, using soapy water mite washes28, with the rate for August 2017 (when the larvae
and adults for the experiments were collected) used in the analyses (Supplementary Fig.1a).
Preparation and optimization of virus material. e DWV and ABPV inocula used in each oral infec-
tion experiment was prepared previously23 in accordance with standard pupal virus propagation procedures13.
In brief, the inocula were prepared by propagating reference stocks of DWV-A and ABPV each in y white
eyed pupae from varroa-free colonies23. Each pupae was injected with 1 micro litre of a 1/10,000 dilution of
puried concentrated virus stock (equivalent to about 103 virus genome copies/bee)13. From these 50 pupae, a
claried crude extract was made by homogenizing the pupae in a blender with 10mL 0.5M Phosphate Buer,
pH 8.0 (DWV) or 10mL 0.01M Phosphate Buer, pH 7.0 (ABPV),and stored in 50μl aliquots at – 80°C13. ese
crude extracts were used for the virus infection experiments. e virological composition of the propagated
virus stocks has been described previously19 and was determined using RT-qPCR assays for seven common bee
viruses that can be propagated through injection13: DWV-A, DWV-B, ABPV, IAPV, KBV, sacbrood virus (SBV)
and black queen cell virus (BQCV).
e infectivity of the crude extracts was tested previously in optimization experiments23 to identify the opti-
mum dose for experimentation that did not cause larvae or adult bee mortality before 96h post inoculation (hpi).
is dose selection criteria was used so that early (non-lethal) virus infectivity dynamics could be studied, as well
as possible subsequent dierential mortality between mite-resistant and mite-susceptible bees. e optimum
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single inoculation dose for larvae was determined to be ~ 1.5 × 108 and ~ 6.0 × 108 DWV genome equivalents for
larvae and adults respectively, with the corresponding gures for ABPV inoculation ~ 5.4 × 107 and ~ 2.1 × 108
ABPV genome equivalents, as determined by RT-qPCR analysis of the crude extracts. ese levels are consistent
with previous estimates of the infectious doses for these viruses13, 15, 23, 29, 30.
Experimental design. e infection experiments were conducted separately on newly emerged adult bees
and on newly hatched larvae. Each infection experiment consisted a single infection time-course for bees from
four dierent colonies from each population. With a few exceptions, the same colonies were used for both the
larval and adult experiments. Each infection trial consisted of one cohort of DWV-inoculated bees, one cohort
of ABPV-inoculated bees and one cohort of non-inoculated control bees. From each cohort of bees in each infec-
tion trial, adult bees and larvae were sampled at 0, 6, 24, 48, and 72h post inoculation (hpi) representing the
time-course. e inoculation strategy consisted of feeding bees with a single infectious dose for a short period
followed by non-contaminated food for the remainder of the time course, in order to ensure that any increase in
virus titres through the time course represented a newly established infection rather than a passive accumula-
tion of virus inoculum. e non-inoculated cohorts received food containing crude extract from non-inoculated
pupae.
In‑vitro larval infection experiments. e larval infection experiments were conducted on newly
hatched larvae from 4 colonies from each of the four mite-resistant populations (Norwegian, Swedish, Dutch,
French) and the mite-susceptible Control population. Larvae of similar age were obtained by conning the
queens of the experimental colonies to a single frame for 24h for egg-laying. First instar larvae (between 24
and 36h old) were transferred into individual wells of 48-well tissue culture plates (Falcon™ Polystyrene Micro-
plates) following standard larval rearing procedures23, 31, 32. e larvae were pre-incubated for 24h at 35°C with
a relative humidity of 96%, aer which all dead and excess larvae were removed, such that 48 living larvae were
retained for the infection experiment. e viable larvae were then fed with larval food. For the larvae cohorts to
be inoculated with virus, the larval food was mixed with the optimum single infectious dose of DWV or ABPV,
as determined above. e larvae were fed daily according to established protocols32. At each hpi-sampling point
during the experimental time-course (see above), four live larvae from each infection cohort were collected in
microcentrifuge tubes and stored at − 20°C until further analysis33.
Adult bee cage infection experiments. e adult infection experiments were conducted on newly
hatched adult bees from (generally) the same 4 colonies used for the larvae infection experiments from each of
the ve populations. e bees were hatched on caged frames inside an incubator at a constant 35°C temperature
and 96% relative humidity34. e level of varroa infestation of each frame was assessed on a four-point ordered
scale (0–4 for none, low, medium and high) based on the number of mites encountered in the cage during the
emergence of the adults (Supplementary Fig.1b). For each inoculation trial, cohorts of y newly emerged
adults from each colony were placed in separate Lyson queen cages (Łyson, Klecza Dolna, Poland), and fed the
optimum DWV and ABPV inoculation dose (as described above) in 2mL Bifor® (Nordic Sugar A/S, Copenha-
gen, Denmark) over a 24-h period, with uninoculated bees receiving just Bifor®. Aer inoculation, all cohorts of
bees were fed unadulterated Bifor® adlibitum for the remainder of the time-course. On each sampling occasion
(see above) and for each infection cohort, ve live bees were sampled. To study the survival rate of each popula-
tion to virus infections, all dead bees were also counted and removed at each sampling time point. e experi-
ments continued for six days (144 hpi), aer which the number of dead and surviving bees were counted. All the
sampled live and dead bees were stored at − 20°C until further analysis.
Sample processing and RT‑qPCR assays. Each experimental time-course sample, containing either
4 larvae or 5 adult bees, was placed in a mesh bag and ground to powder using liquid nitrogen and a pestle.
A primary homogenate was produced by adding 200μl/bee sterile water to each ground sample and mixing
vigorously33. Total RNA was extracted from 100μl of this homogenate by a QiaCube robot following the RNAe-
asy protocol for plants (Qiagen). e RNA was eluted in 50-μl RNase-free water, the RNA concentration was
estimated by NanoDrop and the puried RNA was stored at − 80°C until further processing.
e amounts of DWV and ABPV RNA, as well as RP49 mRNA (a honeybee internal reference gene com-
monly used for normalizing between-sample dierences in RNA quantity and quality13) were determined using
reverse transcription quantitative PCR (RT-qPCR), using the iScript One Step RT-PCR kit (Bio-Rad) with SYBR
Green as the detection chemistry and the Bio-Rad CFX connect thermocycler. e reactions were performed
in 20μl volumes containing 0.2μM of the forward and the reverse primers, 3μl RNA, 10μl SYBR Green RTmix
and 0.4μl of iScript reverse transcriptase, with the following cycling prole: 10min at 50°C for cDNA synthesis,
5min at 95°C for inactivation of the reverse transcriptase following 40 cycles of 10s. at 95°C for denaturation
and 30s. at 58°C for annealing/extension and data collection. Amplication was followed immediately by a
Melting Curve analysis to conrm the identity of the amplication products, by incubating at 60s: 95°C, 60s
65°C and uorescence reading at 0.5°C increments between 65 and 95°C. Included in each qPCR run was
a ten-fold dilution series of known amounts of each target, for absolute quantication. All assays were run in
duplicate, with the average Cq value retained for analysis. e qPCR data were rst screened for the presence
of secondary RT-PCR products through visual inspection of the Melting Curve (MC) analyses. Aer the visual
inspection, the average Cq values were converted to Standard Quantity (SQ) values through use of the external
calibration curves established by the ten-fold dilution series for each target. ese data were then multiplied by
the various dilution factors throughout the methodology to estimate the copy number of each target per bee.
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e DWV and ABPV values for each sample were then normalized using their corresponding RP49 values, to
correct for sample-specic dierences in the quality and quantity of RNA35, 36.
Statistical analyses. e normalized virus titres per individual bee were log10 transformed and ana-
lysed with General Linear Mixed Models using the R soware37. e titres of either of the two viruses (DWV,
ABPV) as recorded for the infection experiments for either of the two life stages (larvae, adults) were used as
the response variables, analysed separately. In each of these four separate analyses the models (function lmer
within the lmerTest package38) tested the degree to which the virus titres depended on the Population of origin,
the Inoculation treatment (DWV, ABPV, no virus) or the phoretic Varroa infestation rate of the colonies pro-
viding the experimental bees, and their interactions. e apiary location of the colonies and the dierent post-
inoculation sampling time-points during the time-courses were included as Gaussian random eects to account
for, respectively, any apiary-specic variability between the colonies used and the repeated measure structure
associated with sampling the same group of bees progressively during the time-course. e distribution of the
residuals and the homogeneity of variances was checked visually to conrm compliance with the assumptions
for linear models using a Gaussian-distributed response variable39. Pairwise comparisons (using the function
glht and cld from the mulcomp package40) among all combinations of Population and Inoculation treatment
were calculated from these initial models. In order to evaluate the importance of each of these predictors and
their interaction, non-signicant terms were removed in a backwards model selection (using the step function
in the lmerTest package) until the minimal adequate model was obtained.
To examine dierences in individual bee mortality probability between varroa-susceptible and varroa-resist-
ant populations, we implemented a time-to-event (or ‘survival’) analysis using a Cox proportional hazards regres-
sion model41. Here we estimated the probability that an individual bee died relative to time post-inoculation.
ese probabilities were compared between the dierent populations, in particular with pairwise comparisons
between the varroa-susceptible Control and each of the Dutch, French, Norwegian and Swedish varroa-resistant
populations. e analyses were run separately for DWV-inoculated, ABPV-inoculated and non-inoculated adult
bees. For each of these three virus inoculation experiments, we followed the fate of 600 individual bees (30 bees
from each colony × 4 replicate colonies per population × 5 population) and recorded their survival or mortality
at 6, 24, 48, 72, 96 and 144h post-inoculation (a total of 3600 potential individual bees observations). Because
the colonies were located at four dierent apiaries, with dierent historical and environmental backgrounds
(see “Origin of honeybee colonies), we included ‘location’ as a random eect with a so-called ‘frailty model’42.
Without this random eect, the Schoenfeld analysis of the residuals43 indicated some violation of the proportional
hazards assumptions. For all Cox Proportional Hazard analyses we included two covariates to explain dierences
in mortality: the population origin (i.e. Control, Dutch, French, Norwegian and Swedish) and the colony-level
phoretic varroa infestation rate (mites per adult bee) for each colony during August 2017, when the experiments
took place, to control for any confounding eects that dierent varroa mite loads would contribute to individual
bee mortality. e models converged readily (11, 12 and 14 outer iterations; 28, 31 and 35 Newton–Raphson
iterations) and could explain the data with a very high degree of condence, as assessed by a Likelihood ratio
test (Supplementary Table1). e analyses were conducted in R37 using the ‘coxme’ package44.
Results
Virus infection time courses. e virus infection time courses for the two viruses were very similar to
those reported previously19. A graphical summary of the raw data is shown in Supplementary Fig.2. For the
DWV infection experiments in larvae there was large increase in DWV titre between the pre-inoculation and
the rst post-inoculation time-point (at 6 hpi), followed by slight increases for the remainder of the time-course,
in all ve populations. ere were generally no DWV titre increases in either the non-inoculated control series
or the ABPV-inoculated series, except for the French population and, to a lesser degree, the Dutch population.
e DWV infection experiments in adults were compromised by the very high background DWV titres in the
populations, similar to what we observed previously19, making it impossible to demonstrate successful infec-
tion either through comparing pre- and post-inoculation samples or through titre increases with time, since
such increases were observed equally in all populations for the DWV-inoculated, the ABPV-inoculated and the
non-inoculated series. It is only through the eect of DWV inoculation on adult mortality (see later) that we
know that the inoculum actually had an eect on the bees. For the ABPV infection time-course experiments
there is clearer evidence of a slow progressive increase in ABPV titres, in both larvae and adult bees, and for all
populations, suggesting that the inoculation resulted in an active infection. ere was no great dierence in the
background ABPV titres between the DWV-inoculated and non-inoculated individuals for either the larval or
adult infection experiment. For the adult experiment there was a slight increase in these background ABPV titres
over time, whereas for the larval experiment there was not.
Virus susceptibility. Although both inoculated and background virus titres tended to increase slightly over
time in both the larval and adult experiments and for all populations, oen enough to suspect that these repre-
sented active infections, these increases were not large enough with respect to the replicate error variance to be
signicant. For the remainder of the analyses therefore the values from the time-course were pooled, eectively
treating time post-inoculation as a random factor in the GLMM analyses. is meant that the data from the
entire time-course were compressed into a single value, which can be taken as a measure of the overall suscep-
tibility of the population to DWV or ABPV infection over the entire time-course, as well as the susceptibility
to background DWV or ABPV infections due to inoculation with the alternate virus (Fig.1; Supplementary
Table1). ese estimates include a correction for the independent eect of the colony-level varroa infestation
rates on the susceptibility to oral DWV or ABPV inoculation (Table1; Supplementary Fig.3).
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Figure1. Graphical representation of the DWV (top panels) and ABPV (bottom panels) titres pooled across
the entire larval (le panels) and adult (right panels) infection time-course experiments for the virus-susceptible
Control population (grey) and the Dutch (yellow), French (green), Norwegian (blue) and Swedish (red)
varroa-resistant populations. e dark bars concern the data where the PCR assay detects the inoculated virus.
e other bars concern the data where the PCR assay detects background virus, either in the non-inoculated
control series (light bars) or the series inoculated with the alternate virus (medium bars). e mean values were
estimated using Generalized Linear Mixed Models with time and colony as a random factor and colony-level
phoretic varroa infestation rate as an explanatory factor. e error bars represent the 95% condence interval
on the estimate. e letters are used to identify dierences between population and inoculation combinations
within each virus-life stage combination.
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For DWV titres as the response variable, there is a clear dierence between the ve populations in susceptibil-
ity of the larvae to oral inoculation with DWV, with the varroa-susceptible Control population colonies consider-
ably more susceptible than the varroa-resistant Dutch, French, Norwegian and (especially) Swedish populations
(Fig.1; Table1). ere is also divergence in background DWV susceptibility between the ve populations, in the
ABPV-inoculated or non-inoculated larvae, with the Dutch and Norwegian populations having noticeably lower
background DWV infection than the Swedish, French and Control populations. However, for each population
there was no signicant dierence in DWV susceptibility between ABPV-inoculated or non-inoculated larvae, as
we also found previously23. As indicated above, the adult infection experiment was dominated by the extremely
high background DWV levels, making it impossible to detect any additional increase in DWV titre due to oral
inoculation. Consequently, the DWV titres for the DWV-inoculated, ABPV-inoculated and non-inoculated bees
were similar within each population, with no eect of the dierent virus inoculation treatments (Fig.1; Table1).
e only signicant dierence is between the dierent populations, with the highest DWV titres in the French
bees, followed by the Dutch, Control, Swedish and Norwegian bees (Fig.1; Table1).
e general pattern seen for the DWV titres as response variable was repeated with ABPV titres as the
response variable, but with some key dierences. Again, the inoculation treatment was highly signicant, both
in larvae and in adults, as shown by the dierence between the ABPV titres in the ABPV-inoculated series com-
pared to the DWV-inoculated and non-inoculated series. Again there is no dierence in ABPV titres between
non-inoculated and DWV-inoculated larvae for each population. For the larval inoculation, there was highly
signicant dierence between the populations in ABPV susceptibility (Table1), although this eect may be more
due to the dierences in the background ABPV titres in the DWV-inoculated and non-inoculated larvae than
due to the dierences between populations in the ABPV-inoculated larvae (Fig.1). Again, the varroa-susceptible
Control population appears to be most susceptible to inoculated ABPV, followed by the Norwegian, Swedish,
Dutch and French populations. For the adult bees, the Control population was again most sensitive to ABPV
inoculation, with the Dutch, French, Norwegian and Swedish populations progressively less susceptible. e
background ABPV titres in the adult bees were relatively similar for all populations, with no dierence between
non-inoculated and DWV-inoculated bees. e general uniformity between the populations in especially the
background ABPV titres, coupled with the relatively high error variance, means that there was no signicant
dierence overall between the populations in ABPV susceptibility (Table1).
e phoretic varroa infestation rate of the colonies consistently had a highly signicant eect on the ABPV
and DWV susceptibility of the colonies (Table1). e relationship between colony-level varroa infestation and
DWV or ABPV susceptibility was largely neutral for the larval inoculation experiments (Supplementary Fig.3),
which is not surprising since varroa has no direct interaction with young brood. Any eects of varroa infestation
Table 1. Analysis-of-deviance tables (Type III test) for the four linear mixed models investigating the eect of
honeybee population (Control, Dutch, French, Norwegian, Swedish) and inoculation treatment (DWV, ABPV,
non-inoculated) on the titres of DWV and ABPV in larvae and adults. Non-signicant terms (italicized)
were removed stepwise, starting with the Population:Virus interaction term, until the nal minimal adequate
model was obtained. Indicated are the Virus studied; the bee Life stage investigated in the inoculation time-
course; the explanatory Variable analysed (Virus inoculation treatment; honeybee Population of origin;
their Population:Virus interaction and the eect of the colony-level phoretic varroa infestation rate for each
colony (see Supplementary Fig.3 for the direction of the eect)); the variation for each variable; the Degrees
of Freedom associated with the target variable (DFv) and the combined error variance (DFe), derived from the
replicate colonies and the multiple post-inoculation sampling time-points; the F-value representing the ratio
between the target MS and the error variance, and the probability (P) of obtaining this F-value by chance,
given the DFv and DFe. Signicance was set at p < 0.01, with non-signicant items shown in italics.
Virus Life stage Var iabl e Variation DFv; DFeF-value P
DWV Larvae
Population 25.47 4; 219.31 4.57 0.001
Virus 452.22 2; 224.02 81.22 < 0.001
Population:Virus 12.83 8; 224.66 2.30 0.022
varroa 60.23 1; 227.23 10.82 0.001
DWV Adult
Population 17.64 4; 260.36 8.32 < 0.001
Virus 0.37 2; 259.86 0.17 0.842
Population:Virus 1.82 8; 248.68 0.85 0.560
varroa 165.42 1; 261.64 77.99 < 0.001
ABPV Larvae
Population 25.01 4; 225.26 7.48 < 0.001
Virus 608.12 2; 224.48 181.86 < 0.001
Population:Virus 12.18 8; 224.25 3.64 0.001
varroa 41.60 1; 227.05 12.44 0.001
ABPV Adult
Population 5.61 4; 233.11 1.58 0.182
Virus 228.15 2; 262.22 63.34 < 0.001
Population:Virus 6.31 8; 249.37 1.82 0.074
varroa 29.45 1; 100.69 8.18 0.005
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on larval susceptibility therefore has mediated indirectly through colony-level processes. However, the emerging
adult bees are directly aected by the colony-level varroa infestation rate (Supplementary Fig.1a,b), since var-
roa reproduces on developing pupae1. Consequently, relationship between colony-level varroa infestation and
virus susceptibility of the adult bees is much more pronounced, with higher infestation rates associated with
greater susceptibility to oral virus infection (Supplementary Fig.3). In all four experiments, the random eects
of apiary location (DWV-larvae: p < 0.01; DWV-adult, ABPV-larvae: p < 0.001; ABPV-adult: p < 0.05) and time
post infection (DWV-larvae: p = 0.06; ABPV-larvae: p < 0.05; DWV-adult, ABPV-adult: p < 0.001) were relevant.
Adult mortality. For the adult infection time-course experiment, dead bees were removed daily from the
experimental cages for six days (144h) post-inoculation. is mortality data was analysed with reference to the
population of origin and the inoculation treatment, using Cox’s Proportional Hazard analyses. Without any
experimental virus inoculation there is no dierence between any of the populations in background mortality
(Fig.2A; Table2), which is an acceptable15–20% over 6days. However, aer experimental DWV or (espe-
cially) ABPV inoculation, adults from the varroa-susceptible Control population are much more likely to die
than bees from any of the varroa-resistant target populations (Fig.2B,C; Table2). For the DWV-inoculated
bees there are also slight dierences in mortality between the dierent varroa-resistant populations, while for
the ABPV-inoculated bees this is less evident. Since the colonies used in these experiments had not received
any varroa control for about 1year (see “Materials and methods”), they had varying rates of colony-level var-
roa infestation at the start of the virus inoculation experiments (Supplementary Fig.1a), which reected the
subjective assessments of varroa infestation for the emerged adult bees emerged (Supplementary Fig.1b). Since
varroa infestation by itself has an enormous eect on post-emergence adult survival1, 45, the colony-level var-
roa infestation rates were included in the modelling of adult mortality, to neutralize the eect of the dierential
varroa infestation rates between the colonies and populations (Supplementary Fig.1) on the primary results,
which are the comparisons between the dierent populations and virus inoculation treatments (Fig.2; Table2).
However, these analyses revealed some interesting and contradictory results. e most revealing variable in
Table2 is the exponentiated coecient (expcoecient) which shows both the size of the eect and the direction,
with values < 1.00 indicating a lower mortality due to the factor, and values > 1.00 a higher mortality. As is also
shown in Fig.2, the bees from each of the varroa-resistant populations had a lower mortality than bees from the
Control population in both the DWV-inoculation (blue) and ABPV-inoculation (orange) experiments (expo-
nentiated coecients < 1.00 throughout) while in the absence of experimental virus inoculation (grey), the bees
from the Control colonies actually survived better than those from the varroa-resistant control populations
(exponentiated coecients > 1.00 throughout). By the same logic we see that for the non-inoculated bees and
ABPV-inoculated bees, colony-level varroa infestation had, as expected, a huge and highly signicant negative
eect on survival, with very large exponentiated coecients. However, for DWV-inoculated adults, varroa had a
net positive eect on survival (exponentiated coecient < 1.00), although the results were a little diuse, as indi-
cated by the modest χ2 and associated probability (p = 0.02). is result is due to a couple of (Control) colonies
with very large varroa infestation rates that signicantly overperformed with respect to expectation, together
with several low-infestation colonies that underperformed. e interesting result is that the same bees, with the
same infestation rates, reacted so very dierently depending on whether they were inoculated with DWV, with
ABPV or not inoculated at all. Obviously, non-inoculated bees survive much better than DWV-inoculated or
ABPV-inoculated bees, with or without varroa (Fig.2), but the absence of this interactive eect between varroa
and oral DWV inoculation may be one more factor explaining why DWV consistently emerges as the primary
varroa-associated virus in apiculture5, 4648. e nal factor to aect mortality is the apiary origin of the colonies,
with a clear and consistent ranking of the four apiaries for all three analyses, with the bees from colonies in the
Figure2. Cox Proportional Hazard curves for the Dutch, French, Norwegian and Swedish varroa-resistant
honeybee populations and the varroa-susceptible Control population for the non-inoculated (A), DWV-
inoculated (B) and ABPV-inoculated (C) adult bee virus infection experiments. e shaded areas represent
the 95% condence intervals for the proportional hazard lines, based on the data from four replicate honeybee
colonies for each population. e eects of varroa infestation and apiary origin of the colonies have been
accounted for in the models.
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‘Enköping’ and ‘Rimbo’ apiaries having better survival than expected, and those in the ‘Sigtuna’ and ‘Funbo’
apiaries lower survival than expected, independent of the virus treatment (Table2).
Discussion
Honeybee colony health and pathogen abundance and prevelence is regulated by a variety of factors including
season, geographical location, colony dynamics, pathogen strain and the individual and colony level immune
responses49. erefore, laboratory studies, like this one, that remove these confounding factors, are ideal for
isolating and exploring the mechanisms involved in the interactions between viruses and bees, especially at
individual level. Here we investigated the susceptibility of young larvae and emerging adults from four distinct
varroa-resistant honeybee populations to oral inoculation with two major honeybee viruses, DWV and ABPV,
relative to those of a varroa-susceptible Control population, as well as the mortality of the adult bees from the
experiments. We also looked at the inuence of confounding factors on virus susceptibility and adult mortality,
such as the colony-level varroa infestation rates and the apiary origins of the colonies supplying the bees for
the experiments. e results can be summarised as follows. First, oral inoculation generally elevated virus titres
above pre-inoculum titres plus passive acquisition of the inoculum, and increased slightly with time, indicating
Table 2. Regression coecients ± standard errors, the exponentiated value of these coecients (expcoecient),
the Chi-squared value (χ2), degrees of freedom (DF) and the associated probability of obtaining these χ2
values by chance (P) from Cox proportional hazards models describing the probability of mortality for
individual bees aer being inoculated (Inoculum) with DWV or ABPV, as well as for non-inoculated bees
(none). e main Factors explored for explaining the variation in the data are dierences between the varroa-
susceptible Control population and each of the four varroa-resistant populations (Dutch, French, Norwegian
and Swedish), as well as the eect on adult survival of the phoretic varroa infestation rates of the colonies when
the experimental adult bees were sampled (Varroa) and the apiary location of the colonies at SLU’s Lövsta
Research Station (Frailty), with exponentiated coecients for each of the four apiaries (‘Enköping, ‘Funbo,
‘Rimbo’ and ‘Sigtuna’). e exponentiated coecients converts the regression coecients to a proportional
scale, where values < 1.00 represent a lower mortality due to the factor, and values > 1.00 a higher mortality.
e eects of the four apiaries concern relative internal dierences, with the exponentiated coecients totaling
4.00 in each analysis.
Inoculum Factor Coecient ± SE expCoecient χ2DF P
DWV
Dutch vs Control − 1.22 ± 0.24 0.30 25.34 1.00 < 0.001
French vs Control − 1.18 ± 0.24 0.31 24.42 1.00 < 0.001
Norwegian vs Control − 1.57 ± 0.29 0.21 29.52 1.00 < 0.001
Swedish vs Control − 0.84 ± 0.25 0.43 10.88 1.00 < 0.001
Var roa − 1.58 ± 0.72 0.21 4.82 1.00 0.028
Frailty (apiary eect) 75.59 2.91 < 0.001
Enköping 0.22
Funbo 1.81
Rimbo 0.51
Sigtuna 1.46
ABPV
Dutch vs Control − 0.89 ± 0.25 0.41 13.19 1.00 < 0.001
French vs Control − 1.29 ± 0.27 0.28 23.08 1.00 < 0.001
Norwegian vs Control − 1.05 ± 0.28 0.35 13.68 1.00 < 0.001
Swedish vs Control − 1.50 ± 0.29 0.22 26.33 1.00 < 0.001
Var roa 3.19 ± 0.70 24.27 20.76 1.00 < 0.001
Frailty (apiary eect) 63.92 2.90 < 0.001
Enköping 0.17
Funbo 1.81
Rimbo 0.51
Sigtuna 1.51
None
Dutch vs Control 1.25 ± 0.46 3.48 7.23 1.00 0.007
French vs Control 0.48 ± 0.52 1.62 0.84 1.00 0.360
Norwegian vs Control 0.46 ± 0.54 1.58 0.71 1.00 0.400
Swedish vs Control 1.10 ± 0.48 3.01 5.26 1.00 0.022
Var roa 4.19 ± 1.26 65.94 10.99 1.00 < 0.001
Frailty (apiary eect) 18.48 2.68 < 0.001
Enköping 0.21
Funbo 1.76
Rimbo 0.96
Sigtuna 1.07
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that the inoculation resulted in infection. e exception is the inoculation of the adult bees with DWV, where
the exceptionally high background DWV levels precluded any conclusive evidence of infection. Second, there
were clear dierences between the dierent populations in susceptibility to DWV and ABPV infection, either
through inoculation and/or as background infections, both before and aer correcting for the eects of varroa
and apiary origin, with the Control population usually displaying the highest susceptibility. ird, inoculation
with one virus generally did not aect the background levels of the other virus relative to the non-inoculated
control, for any of the populations or inoculation experiments, similar to what we found previously23. Fourth,
adult bees from the varroa-susceptible Control population had much higher mortality aer oral DWV or ABPV
inoculation than bees from any of the four varroa-resistant populations, while there was no major dierence
between non-inoculated bees from the various populations. Clearly, infection with these viruses is tolerated
much better by the varroa-resistant bees than the varroa-susceptible bees, independent of any dierences in virus
titres. Fih, varroa infestation and apiary origin have signicant eects on virus susceptibility and mortality in
adult bees. Colony-level varroa infestation is associated with higher susceptibility of adult bees to both DWV and
ABPV infection, and with much higher mortality of both non-inoculated and ABPV-inoculated bees, but not of
DWV-inoculated bees. is absence of an independent eect of varroa infestation on adult bee mortality in only
DWV-inoculated bees is particularly interesting in light of the many other exceptional features of DWV that
are peculiarly adaptive to optimal co-existence with varroa2, 2325, 48, and that have elevated DWV from relative
obscurity to global prominence in the wake of varroa8, 47, 50. Apiary eects are a well-known source of error in
honeybee research, particularly for colony-level experiments. Here we show that such landscape-level dierences
can also aect individual level laboratory results, serving both as a caution for those contemplating laboratory
studies without due regard for the origin of the bees and as a promise for investigating in greater molecular detail
the mechanisms linking the inuence of the environment on honeybee molecular health51.
Another consistent feature of these experiments is the large variation in the background virus levels between
the ve populations in all experiments, which contributed majorly to the signicance of the population eect
in the analyses. e only experimental dierence between these colonies was the origin of the queens during
establishment in 2016, aer which the colonies developed according to the local environmental conditions in
the apiaries and the colony development characteristics associated with the genotype of the queen. e colonies
of the ve populations were distributed systematically and evenly among the four apiaries; three colonies of each
population to each apiary, with only these colonies present in each apiary. e four apiaries were located near the
center of the extensive SLU Agricultural Research Station at Lövsta, several kilometres from any surrounding bee
colonies. In the absence of any compelling environmental explanation, the conclusion is that these background
virus levels are also a characteristic trait of these colonies and populations, although the signicance of this is
as yet unclear. e dierences are particularly striking for the larvae inoculation experiments. Both DWV and
ABPV can be vertically transmitted, from queen through her eggs to the resulting progeny7, 8, 10, 11, 52, and from
there to the rest of the colony through larval care and social interactions53, 54. Any systematic dierence between
queens in the level of virus infection52, 55 and/or the eciency of vertical transmission would lead to dierences
in ‘background’ DWV or ABPV infection in young, newly hatched larvae, such as used in these experiments.
ABPV is rare in Sweden and Norway56, very common in France4 and moderately common in e Netherlands57,
which does not match very well with the relative background ABPV levels in the larvae from these populations.
Furthermore, a large survey of virgin and mated queens from southern France showed no ABPV infection of
the queen ovaries, in contrast to the high frequency (and levels) of DWV-A, DWV-B and BQCV infection.
erefore, even if the queens did vertically transmit viruses from their geographic origin to the larvae used in
these experiments, it would not fully explain all the dierences in the larval background virus levels between the
populations. It is therefore possible that some of these dierences at least are related to the genetic background
of the populations, either directly, at individual bee level, or as mediated through colony-level processes that
also dier between the populations. For the adult experiments the DWV background levels were so high as to
preclude any conclusion of the infectivity of the DWV inoculum in these experiments. Again there are very clear
and consistent dierences between the populations in the background DWV levels, suggesting a corresponding
dierence between the populations in background tolerance or resistance to DWV infection. e background
ABPV levels in the adult bees were more uniform between the populations, suggesting no such dierential
tolerance-resistance between the populations for background ABPV infection.
e most signicant conclusion from these ve major results is that adult bees from naturally varroa-resistant
bee populations are much more tolerant to oral DWV or ABPV infection than bees from regular varroa-sus-
ceptible control populations, as shown both here and, independently, in our earlier studies23, 24. e most likely
explanation for this elevated tolerance to virus infections is that the natural adaptation of these populations to
uncontrolled varroa infestation included a degree of tolerance to virus infections in addition to their already well
established genetically adapted varroa-resistant traits19, 20. Recent studies on the naturally adapted mite-resistant
honeybee population on Gotland, Sweden, have demonstrated adapted host tolerance to virus infections at both
levels of honeybee social and biological organization: the colony24 and the individual bee23. Apart from this, few
studies have explored host-adaptations to virus infections58, 59. e populations in this study provide the oppor-
tunity to explore possible tolerance or resistance mechanisms that have arose in populations through natural
selection having been exposed to long-lasting likely subclinical virus infections inducing a substantial selective
pressure. Virus tolerance and resisance could provide a new and exciting avenue for breeding healthier bees.
Tolerance is a highly eective mechanistic response to disease60. Unlike resistance mechanisms, tolerance adapta-
tions do not inict harm on the parasite and is therefore expected to x in the population rather than causing an
open-ended antagonistic coevolution, as is the case with resistance evolution60. In addition to uncontrolled mite
infestations and high virus levels, environmental inuences would have also shaped the genetic adaptations in
responses to these parasites61. e dierent geographical origins of the populations used in this study, ranging
from Scandinavia to South of France, have dramatically dierent environmental conditions such as the season
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length, temperature, and oral resources. e possible inuence of such environmental factors on the nature
of the adaptive process and the consequences for the dierent varroa-resistant and virus-tolerant traits of these
populations is a topic of current and future research.
e varroa infestation rates in the colonies that were used for these experiments signicantly increased the
probability of adult bee mortality regardless of inoculation with either virus. is is by itself not surprising, since
varroa infestation causes a range of physical and physiological eects for both individual bees and the colony. Not
only does varroa mite parasitization directly aects the longevity of adult bees45, it also acts as both a mechanical
and biological vector of DWV and other viruses53, 62, 63, signicantly enhancing the epidemiological potential and
lethality of virus infections15. Varroa mite parasitism also comprimises the honeybee host’s immune response to
virus infections by suppressing the expression of immune response related genes64 and increasing viral titres in
the bee, both of which reduce adult bee survivorship and colony tness65, 66.
e interactions between the mites, the viruses and the honeybee molecular antiviral defense mechanisms
and immune functions is a subject of considerable current research6770, but also with considerable gaps in
knowledge and understanding71. e large set of precise samples generated during this study together with the
extensive metadata relating to the genetic and environmental background of the colonies provides a rich source
of material to address these knowledge gaps from a number of perspectives.
Data availability
e datasets generated during and/or analysed during the current study are available from the corresponding
author on reasonable request.
Received: 17 February 2021; Accepted: 28 May 2021
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Acknowledgements
e authors wish to thank Emilia Semberg, Eva Forsgren and various eld- and lab-assistants for their support
during this project. ese experiments were funded by FORMAS grants awarded to JM and BL (Dnr. 2013-1225)
and to BL (Dnr. 2016-00481) and by the Ricola Foundation Nature & Culture (PN).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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Author contributions
Conceptualization: B.L., S.T., J.M.; methodology: S.T., J.M.; validation: B.L., S.T., J.M.; investigation: S.T.; formal
analysis: J.S., M.L.; data curation: S.T., J.M.; writing: B.L., S.T., J.M.; editing: all; visualization: J.M., M.L.; resources:
B.L., T.B., B.D., Y.L., J.M.; supervision: B.L., J.M.; project administration: J.M.; funding acquisition: B.L., J.M.,
P.N. All authors read and approved the nal manuscript.
Funding
Open access funding provided by Swedish University of Agricultural Sciences.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 021- 91686-2.
Correspondence and requests for materials should be addressed to B.L.
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... Furthermore, the genotypic differences underlying miteresistant behaviors may be associated with reduced levels of mite-vectored viruses such as DWV (Locke et al., 2014;Khongphinitbunjong et al., 2016;de Guzman et al., 2017;Mendoza et al., 2020;Weaver et al., 2021;O'Shea-Wheller et al., 2022). Such bee genotype × DWV interactions may be the result of enhanced virus resistance or tolerance in certain bee stocks, with prior data indicating a greater potential for tolerance (Strauss et al., 2013;Locke et al., 2014;Khongphinitbunjong et al., 2015;Khongphinitbunjong et al., 2016;Thaduri et al., 2018;Thaduri et al., 2019;Locke et al., 2021). Resistance is the ability of the bee to prevent an infection from establishing or increasing after exposure, while tolerance is the ability of the individual to maintain health and functionality (i.e., reduced symptoms), even while having an infection (Locke et al., 2014;Mordecai et al., 2016a;Burgan et al., 2018). ...
... For instance, hygienic bees may have higher exposure rates to DWV than nonhygienic bees since hygienic bees exhibit increased rates of cannibalism on mite-infested pupae (Posada-Florez et al., 2021). When we considered adult emergence time and DWV symptom presence and severity, bee stocks appeared to differ in their tolerance to DWV; though these results were not necessarily consistent with stocks bred for mite resistance (Khongphinitbunjong et al., 2016;Locke et al., 2021). In the multivariate and classification tree analyses (Figures 7, 8), the Russian stock most consistently grouped with Carniolan as being DWV-tolerant (high level of the virus with fewer symptoms and/ or lower severity), while Pol-Line grouped with Italian and Saskatraz as more DWV susceptible, particularly when considering symptom severity (Dim.1 axis on Figures 8A,B). ...
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... Differences in defense mechanisms between host species may result in one species coping better with infections than others and/or transmitting a disproportional amount of pathogens [11]. For bee viruses, studies on viral defense strategies on individual level scarce are mostly investigated in conjunction with other factors, such as nutrition or pesticide exposure, which complicates the interpretation of 'pure' viral tolerance or resistance [12][13][14]. There are ample prevalence studies that report links with the viral prevalence in wild bees and the presence of honeybees [15][16][17][18]. ...
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... SBV also interferes with the replication of DWV 40 , ABPV 61 , and possibly other viruses 62 ; is a significant contributing factor to DWV-related virulence and mortality 60 in controlled infection experiments, and may furthermore also affect varroa behaviour 49 . Interference between bee viruses appears to be more evident when the viruses are injected 42,63 , compared to oral inoculation 50,51,61 . These are all significant traits for natural varroa resistance, affecting both varroa and viruses 4,20,64 . ...
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... The veterinary drugs whose residues were identified in the present study were DMF (N-2,4-Dimethylphenyl-formamide) and amitraz (total). Amitraz is used in apiaries against parasitic mites Varroa destructor, which are carriers of pathogenic viruses such as Acute Bee Paralysis Virus (ABPV) and Deformed Wing Virus (DWV) [32]. O'Neal et al. [33], however, showed that Amitraz has certain limitations, because exposure to this compound can adversely affect bees' resistance to viral infections. ...
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... Several studies have demonstrated that viral titers in A. mellifera are correlated with the titers in other bee species in the community (Fürst et al., 2014, McMahon et al., 2015. The amplification of DWV in A. mellifera via Varroa mites has been demonstrated to result in tolerance to DWV (Hinshaw et al., 2021, Locke et al., 2021 and high DWV intensity in A. mellifera colonies, and has been found to significantly increase the abundance of DWV among other pollinating insects (Martin and Brettell, 2019). While we found similarly high DWV intensity in our A. mellifera samples, we did not find comparable copy numbers in E. pruinosa, suggesting that not all bee species act as suitable hosts for DWV. ...
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Managed and wild bee populations are in decline around the globe due to several biotic and abiotic stressors. Pathogenic viruses associated with the Western honey bee (Apis mellifera) have been identified as key contributors to reductions in the number of managed honey bee colonies, and are known to be transmitted to wild bee populations through shared floral resources. However, little is known about the prevalence and intensity of these viruses in wild bee populations, or how bee visitation to flowers impacts viral transmission in agroecosystems. This study surveyed honey bee, bumble bee (Bombus impatiens) and wild squash bee (Eucera (Peponapis) pruinosa) populations in Cucurbita agroecosystems across Pennsylvania (USA) for the prevalence and intensity of five honey bee viruses: acute bee paralysis virus (ABPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and slow bee paralysis virus (SBPV). We investigated the potential role of bee visitation rate to flowers on DWV intensity among species in the pollinator community, with the expectation that increased bee visitation to flowers would increase the opportunity for transmission events between host species. We found that honey bee viruses are highly prevalent but in lower titers in E. pruinosa and B. impatiens than in A. mellifera populations throughout Pennsylvania (USA). DWV was detected in 88% of B. impatiens, 48% of E. pruinosa, and 95% of A. mellifera. IAPV was detected in 5% of B. impatiens and 4% of E. pruinosa, compared to 9% in A. mellifera. KBV was detected in 1% of B. impatiens and 5% of E. pruinosa, compared to 32% in A. mellifera. Our results indicate that DWV titers were not correlated with bee visitation in Cucurbita fields. The potential fitness impacts of these low viral titers detected in E. pruinosa remain to be investigated.
... This independent occurrence of the key traits within colonies across the world could be an example of parallel evolution [27], because while the recapping and removal behaviours predate Varroa, they have been co-opted to control Varroa, recapping is rare trait in mite-naive colonies, but occurs at low and high levels in susceptible and resistant colonies respectively [33,40]. Similarly, other traits such as brood suppression of mite reproduction [48], or DWV tolerance [49,50] may complement those within the framework. There is also likely to be a mite element to resistance which could be illuminated by further studies into the coevolution of A. mellifera and Varroa [51,52]. ...
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The near-globally distributed ecto-parasitic mite of the Apis mellifera honey-bee, Varroa destructor, has formed a lethal association with Deformed wing virus, a once rare and benign RNA virus. In concert, the two have killed millions of wild and managed colonies, particularly across the Northern Hemisphere, forcing the need for regular acaricide application to ensure colony survival. However, despite the short association (in evolutionary terms), a small but increasing number of A. mellifera populations across the globe have been surviving many years without any mite control methods. This long-term survival, or Varroa resistance, is consistently associated with the same suite of traits (recapping, brood removal and reduced mite reproduction) irrespective of location. Here we conduct an analysis of data extracted from 60 papers to illustrate how these traits connect together to explain decades of mite resistance data. We have potentially a unified understanding of natural Varroa resistance that will help the global industry achieve widespread miticide-free beekeeping and indicate how different honeybee populations across four continents have resolved a recent threat using the same suite of behaviours.
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The ectoparasitic mite, Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera) mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. This multi-level system of host-parasite-pathogen interactions makes it difficult to investigate effects of either the mite or the virus on natural host survival. The aim of this study was to remove confounding effects of varroa to examine the role of virus susceptibility in the enhanced survival of a naturally adapted Swedish mite-resistant (MR) honeybee population, relative to mite-susceptible (MS) honeybees. Caged adult bees and laboratory reared larvae, from varroa-free colonies, were inoculated with DWV and ABPV in a series of feeding infection experiments, while control groups received virus-free food. Virus infections were monitored using RT-qPCR assays in individuals sampled over a time course. In both adults and larvae the DWV and ABPV infection dynamics were nearly identical between MR and MS groups, but MS adults suffered significantly higher mortality than MR adults. Results suggest virus tolerance, rather than reduced susceptibility or virus resistance, is an important component of the natural survival of this honeybee population.
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The parasitic mite, Varroa destructor, in combination with the viruses it vectors, is the main cause for global colony losses of the European honeybee, Apis mellifera. However, an isolated honeybee population established in 1999 on the Island of Gotland, Sweden has naturally acquired resistance to the mite, and has survived without mite control treatment for more than 18 years. A recent study has shown that this mite resistant (MR) population also appears to be resistant to Black queen cell virus (BQCV) and Sacbrood virus (SBV) and tolerant to Deformed wing virus (DWV), relative to nearby mite susceptible (MS) honeybee populations. In this study, RNA sequencing was employed to corroborate these previous findings and identify other viral factors that may play a role in the enhanced survival of this mite resistant honeybee population. Two additional honeybee-infecting viruses, Apis rhabdovirus-1 (ARV-1) and Lake Sinai virus (LSV), were identified and near-complete genomes of these two viruses were obtained. Phylogenetic analyses of the assembled virus sequences revealed consistent separation between the MR and MS honeybee populations, although it is unclear whether this is due to pre-existing differences between the viruses in the two populations when they were established, and isolated, or due to virus genetic adaptation towards reduced virulence in the MR population, to promote colony survival. Reverse transcription quantitative polymerase chain reaction(RT-qPCR) analyses show higher ARV and LSV titres in MS colonies compared to MR colonies, gradually increasing from summer to autumn 2009, and reaching maximum titres in the following spring 2010. While the DWV and BQCV titres in MR colonies increased between autumn 2009 and spring 2010, the SBV practically disappeared entirely by spring 2010. Possible explanations for the apparent virus tolerance or resistance in the Gotland mite-resistant honeybee population are discussed.
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Deformed wing virus (DWV) is an important pathogen in a broad range of insects, including honey bees. Concordant with the spread of Varroa, DWV is present in the majority of honey bee colonies and can result in either low-level infections with asymptomatic bees that nonetheless exhibit increased colony loss under stress, or high-level infections with acute effects on bee health and viability. DWV can be transmitted vertically or horizontally and evidence suggests that horizontal transmission via Varroa is associated with acute symptomatic infections. Vertical transmission also occurs and is presumably important for the maintenance of DWV in honey bee populations. To further our understanding the vertical transmission of DWV through queens, we performed three experiments: we studied the quantitative effectiveness of vertical transmission, surveyed the prevalence of successful egg infection under commercial conditions, and distinguished among three possible mechanisms of transmission. We find that queen-infection level predicts the DWV titers in their eggs, although the transmission is not very efficient. Our quantitative assessment of DWV demonstrates that eggs in 1/3 of the colonies are infected with DWV and highly infected eggs are rare in newly-installed spring colonies. Additionally, our results indicate that DWV transmission occurs predominantly by virus adhering to the surface of eggs (transovum) rather than intracellularly. Our combined results suggest that the queens’ DWV vectoring capacity in practice is not as high as its theoretical potential. Thus, DWV transmission by honey bee queens is part of the DWV epidemic with relevant practical implications, which should be further studied.
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Background. Managed, feral and wild populations of European honey bee subspecies, Apis mellifera, are currently facing severe colony losses globally. There is consensus that the ectoparasitic mite Varroa destructor, that switched hosts from the Eastern honey bee Apis cerana to the Western honey bee A. mellifera, is a key factor driving these losses. For >20 years, breeding efforts have not produced European honey bee colonies that can survive infestations without the need for mite control. However, at least three populations of European honey bees have developed this ability by means of natural selection and have been surviving for>10 years without mite treatments. Reduced mite reproductive success has been suggested as a key factor explaining this natural survival. Here, we report a managed A. mellifera population in Norway, that has been naturally surviving consistent V. destructor infestations for >17 years. Methods. Surviving colonies and local susceptible controls were evaluated for mite infestation levels, mite reproductive success and two potential mechanisms explaining colony survival: grooming of adult worker bees and Varroa Sensitive Hygiene (VSH): adult workers specifically detecting and removing mite-infested brood. Results. Mite infestation levels were significantly lower in surviving colonies and mite reproductive success was reduced by 30% when compared to the controls. No significant differences were found between surviving and control colonies for either grooming or VSH. Discussion. Our data confirm that reduced mite reproductive success seems to be a key factor for natural survival of infested A. mellifera colonies. However, neither grooming nor VSH seem to explain colony survival. Instead, other behaviors of the adult bees seem to be sufficient to hinder mite reproductive success, because brood for this experiment was taken from susceptible donor colonies only. To mitigate the global impact of V. destructor, we suggest learning more from nature, i.e., identifying the obviously efficient mechanisms favored by natural selection.
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The parasitic mite Varroa destructor is a serious threat for western honey bee colonies and beekeepers are compelled to control it to keep their colonies healthy. Yet, by controlling varroa no resistance to the parasite can evolve. As a trial, honey bee colonies have been left untreated in isolated locations to allow development of resistance or tolerance to the mite. These colonies developed an ability to live without control measures against varroa, although the traits responsible for this resistance or tolerance are still unclear. Two of these resistant populations have been studied to test the involvement of specific varroa mite targeted hygienic behaviour varroa sensitive hygiene (VSH) in the acquired resistance. Individual mites were manually introduced into just capped brood cells, after which the brood combs were placed in colonies of the two resistant populations and in control colonies in which varroa had always been controlled. We followed the development of the mites, including possible removals. We found that VSH had increased strongly in one of the selections, up to 40% of the infested cells with mites and pupae were removed, but it had decreased in the other selection, compared to the control colonies. Further we could not conclude from our data that VSH only or preferentially targets reproducing mites, leaving non-reproducing mites undisturbed. The different VSH responses between the two selected resistant honey bee populations lead to conclude that more than one mechanism of resistance may evolve in response to the selection pressure by varroa mites.
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Deformed wing virus (DWV) has become the most well-known, widespread, and intensively studied insect pathogen in the world. Although DWV was previously present in honeybee populations, the arrival and global spread of a new vector, the ectoparasitic mite Varroa destructor, has dramatically altered DWV epidemiology. DWV is now the most prevalent virus in honeybees, with a minimum average of 55% of colonies/apiaries infected across 32 countries. Additionally, DWV has been detected in 65 arthropod species spanning eight insect orders and three orders of Arachnida. Here, we describe the significant progress that has been made in elucidating the capsid structure of the virus, understanding its ever-expanding host range, and tracking the constantly evolving DWV genome and formation of recombinants. The construction of molecular clones, working with DWV in cell lines, and the development of immunohistochemistry methods will all help the community to move forward. Identifying the tissues in which DWV variants are replicating and understanding the impact of DWV in non-honeybee hosts are major new goals.
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