Winter survival of individual honey bees and honey bee colonies depends on level of Varroa destructor infestation


van Dooremalen, C.; Gerritsen, L.; Cornelissen, B.; van der Steen, J.J.M.; van Langevelde, F.; Blacquière, T.

Plos One 7(4): E36285

2012


Recent elevated winter loss of honey bee colonies is a major concern. The presence of the mite Varroa destructor in colonies places an important pressure on bee health. V. destructor shortens the lifespan of individual bees, while long lifespan during winter is a primary requirement to survive until the next spring. We investigated in two subsequent years the effects of different levels of V. destructor infestation during the transition from short-lived summer bees to long-lived winter bees on the lifespan of individual bees and the survival of bee colonies during winter. Colonies treated earlier in the season to reduce V. destructor infestation during the development of winter bees were expected to have longer bee lifespan and higher colony survival after winter. Methodology/Principal Findings: Mite infestation was reduced using acaricide treatments during different months (July, August, September, or not treated). We found that the number of capped brood cells decreased drastically between August and November, while at the same time, the lifespan of the bees (marked cohorts) increased indicating the transition to winter bees. Low V. destructor infestation levels before and during the transition to winter bees resulted in an increase in lifespan of bees and higher colony survival compared to colonies that were not treated and that had higher infestation levels. A variety of stress-related factors could have contributed to the variation in longevity and winter survival that we found between years. Conclusions/Significance: This study contributes to theory about the multiple causes for the recent elevated colony losses in honey bees. Our study shows the correlation between long lifespan of winter bees and colony loss in spring. Moreover, we show that colonies treated earlier in the season had reduced V. destructor infestation during the development of winter bees resulting in longer bee lifespan and higher colony survival after winter.

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one
Winter
Survival
of
Individual
Honey
Bees
and
Honey
Bee
Colonies
Depends
on
Level
of
Varroa
destructor
Infestation
Coby
van
Dooremalen'*,
Lonne
Gerritsen',
Bram
Cornelissen',
Jozef
J.
M.
van
der
Steen',
Frank
van
Langevelde
2
,
Tjeerd
Blacquiere'
1
Bees@wur,
Plant
Research
International,
Wageningen,
The
Netherlands,
2
Resource
Ecology
Group,
Wageningen
University,
Wageningen,
The
Netherlands
Abstract
Background:
Recent
elevated
winter
loss
of
honey
bee
colonies
is
a
major
concern.
The
presence
of
the
mite
Varroa
destructor
in
colonies
places
an
important
pressure
on
bee
health.
V.
destructor
shortens
the
lifespan
of
individual
bees,
while
long
lifespan
during
winter
is
a
primary
requirement
to
survive
until
the
next
spring.
We
investigated
in
two
subsequent
years
the
effects
of
different
levels
of
V.
destructor
infestation
during
the
transition
from
short-lived
summer
bees
to
long-lived
winter
bees
on
the
lifespan
of
individual
bees
and
the
survival
of
bee
colonies
during
winter.
Colonies
treated
earlier
in
the
season
to
reduce
V.
destructor
infestation
during
the
development
of
winter
bees
were
expected
to
have
longer
bee
lifespan
and
higher
colony
survival
after
winter.
Methodology/Principal
Findings:
Mite
infestation
was
reduced
using
acaricide
treatments
during
different
months
(July,
August,
September,
or
not
treated).
We
found
that
the
number
of
capped
brood
cells
decreased
drastically
between
August
and
November,
while
at
the
same
time,
the
lifespan
of
the
bees
(marked
cohorts)
increased
indicating
the
transition
to
winter
bees.
Low
V.
destructor
infestation
levels
before
and
during
the
transition
to
winter
bees
resulted
in
an
increase
in
lifespan
of
bees
and
higher
colony
survival
compared
to
colonies
that
were
not
treated
and
that
had
higher
infestation
levels.
A
variety
of
stress-related
factors
could
have
contributed
to
the
variation
in
longevity
and
winter
survival
that
we
found
between
years.
Conclusions/Significance:
This
study
contributes
to
theory
about
the
multiple
causes
for
the
recent
elevated
colony
losses
in
honey
bees.
Our
study
shows
the
correlation
between
long
lifespan
of
winter
bees
and
colony
loss
in
spring.
Moreover,
we
show
that
colonies
treated
earlier
in
the
season
had
reduced
V.
destructor
infestation
during
the
development
of
winter
bees
resulting
in
longer
bee
lifespan
and
higher
colony
survival
after
winter.
Citation:
van
Dooremalen
C,
Gerritsen
L,
Cornelissen
B,
van
der
Steen
JJM,
van
Langevelde
F,
et
al.
(2012)
Winter
Survival
of
Individual
Honey
Bees
and
Honey
Bee
Colonies
Depends
on
Level
of
Varroa
destructor
Infestation.
PLoS
ONE
7(4):
e36285.
do1:10.1371/journal.pone.0036285
Editor:
Mark
F.
Feldlaufer,
United
States
Department
of
Agriculture,
Agriculture
Research
Service,
United
States
of
America
Received
February
7,
2012;
Accepted
March
29,
2012;
Published
April
27,
2012
Copyright:
C
2012
van
Dooremalen
et
al.
This
is
an
open-access
article
distributed
under
the
terms
of
the
Creative
Commons
Attribution
License,
which
permits
unrestricted
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
author
and
source
are
credited.
Funding:
The
project
was
funded
by
Ministry
of
Agriculture,
Nature
Conservation
and
Food
Quality
(LNV)
of
the
Netherlands
and
by
the
European
Union
(project
numbers
NP11/2.1,
NL08/2.1,
BO-06-012-001,
and
BO-12.03-007-001).
The
funders
had
no
role
in
study
design,
data
collection
and
analysis,
decision
to
publish,
or
preparation
of
the
manuscript.
Competing
Interests:
The
authors
have
declared
that
no
competing
interests
exist.
*
E-mail:
coby.vandooremalen@wurn1
Introduction
The
parasitic
mite
Varroa
destructor
is
considered
to
be
one
of
the
main
causes
for
colony
losses
in
honey
bees
(Apis
mellifera
L.)
[1-4].
For
example,
the
total
number
of
honey
producing
colonies
in
the
U.S.
was
reduced
by
1.5±0.7%
(mean
±
s.e.)
per
year
since
the
introduction
of
V.
destructor,
while
the
decrease
per
year
used
to
be
on
average
0.06±0.5%
[1].
This
decline
reflects
the
loss
of
colonies
as
well
as
the
decline
in
number
of
beekeepers
due
to
increased
expenses
and
efforts
needed
to
combat
mite
infestations
[1,5].
Although
there
is
a
general
agreement
that
there
is
no
single
explanation
for
the
extensive
colony
losses,
and
that
interactions
between
different
stresses
are
likely
to
be
involved,
the
presence
of
V.
destructor
in
colonies
places
an
important
pressure
on
bee
health
[2].
V.
destructor
reduces
the
body
weight
and
protein
content
of
individual
bees,
which
is
found
to
shorten
their
lifespan
[6-8].
This
is
especially
important
during
winter
in
temperate
regions
when
long
lifespans
are
a
primary
requirement
to
survive
until
the
next
spring
and
to
nurse
the
first
brood
[7,8].
In
the
temperate
regions,
the
main
colony
losses
due
to
V.
destructor
occur
during
winter
[8].
Nowadays,
winter
losses
are
often
up
to
20%
or
more
in
many
areas
[1,3],
while
twenty
years
ago,
5
to
10%
colony
losses
during
winter
were
common
[2].
In
temperate
regions,
the
number
of
bees
and
brood
in
a
colony
increase
between
April
and
July
and
decrease
between
August
and
October
[9].
However,
the
main
peak
of
the
number
of
bees
and
brood
occur
earlier
in
the
season
than
the
peak
of
mite
abundance
[10,11].
Hence,
mite
infestation
strongly
increases
during
the
period
in
which
the
number
of
bees
and
brood
decrease
[9]
(Figure
1),
resulting
in
an
increasing
number
of
brood
cells
infested
with
V.
destructor
over
time.
It
is
exactly
during
these
months
of
reduction
in
the
number
of
brood
and
rapid
increase
in
mite
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Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
In
fes
ta
t
ion
(m
ites
/
bee
)
35000
Bees
47
30000
Brood
-•-•
25000
Mites
Ta
20000
..........
15000
2
10000
5000
0
4:1)
671
Ct.
m
Figure
1.
Colony
development
for
adult
bees,
worker
brood,
and
Varroa
destructor
mites.
The
daily
number
of
individual
adult
bees
(dotted
line)
and
worker
brood
(striped
line)
was
modelled
over
one
year.
The
number
of
mites
(solid
line)
was
modelled
as
being
the
second
year
of
mite
infestation
with
a
starting
population
of
100
mites
in
the
first
year.
Figure
was
redrawn
from
Martin
[9].
doi:10.1371/journal.pone.0036285.9001
infestation,
that
bees
hatching
from
this
highly
infested
brood
will
become
winter
bees
[9,12].
Adult
bees,
which
are
infested
by
V.
destructor
as
pupae,
do
not
fully
develop
physiological
features
typical
of
long-lived
winter
bees
compared
with
non-infested
workers
[6-8],
making
it
unlikely
for
them
to
survive
until
spring
and
contribute
to
the
build-up
of
the
colony
in
early
spring
[2].
To
date,
however,
the
relation
between
the
lifespan
of
individual
bees
and
colony
losses
for
different
levels
of
V.
destructor
infestation
has
not
been
tested.
When
the
European
honey
bee
(Apis
mellfera)
was
moved
to
areas
where
the
Asian
honey
bee
(A.
ceranae)
was
endemic,
V.
destructor
switched
to
A.
mellfera
and
spread
nearly
worldwide
[2,4].
During
the
first
years
after
its
introduction
in
Europe
and
North
America,
V.
destructor
could
be
easily
controlled
and
be
kept
below
damaging
infestation
levels
by
one
to
two
acaricide
treatments
per
year.
However,
colony
losses
have
recently
started
to
increase
drastically,
despite
the
development
of
more
intensive
acaricide
treatments
[1,2].
Absence,
poor
timing
and
poor
application
of
acaricide
treatment
have
been
reported
to
be
important
causes
for
honey
bee
colony
loss
[13,14].
Especially
when
honey
is
harvested
at
the
end
of
the
bee
season
in
temperate
regions,
acaricide
treatments
are
often
postponed
until
after
the
harvest
to
avoid
residues
in
honey.
However,
the
mite
population
has
often
already
reached
injurious
levels
at
this
time,
namely
the
time
that
winter
bees
are
produced
(Figure
1).
Consequently,
timing
of
acaricide
treatment
in
the
second
half
of
the
summer
season
(July
to
September)
may
thus
affect
winter
survival
of
the
colony.
In
this
study,
the
effect
of
different
levels
of
V.
destructor
infestation
during
the
transition
from
short-lived
summer
bees
to
long-lived
winter
bees
on
the
lifespan
of
individual
bees
and
the
survival
of
bee
colonies
during
winter
was
investigated.
We
manipulated
the
level
of
V.
destructor
infestation
by
reducing
the
number
of
mites
using
acaricide
treatments
at
different
times
(during
July,
August,
September,
or
not
treated),
resulting
in
increased
mite
fall
directly
during
acaricide
treatment
and
in
reduced
V.
destructor
infestation
level
in
the
months
after
this
treatment
(Figure
2
gives
the
expected
infestation
levels
for
different
treatment
moments).
We
expected
a
longer
lifespan
of
bees
in
colonies
treated
earlier
in
the
season,
as
low
infestation
levels
during
the
development
of
winter
bees
should
benefit
the
lifespan
of
these
bees
compared
to
colonies
treated
later
in
the
year
or
not
at
all.
Consequently,
colonies
with
relatively
low
V.
destructor
infestation
during
the
development
of
winter
bees
are
expected
to
have
higher
colony
survival
during
or
after
winter.
The
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Figure
2.
Expected
infestation
levels
of
Varroa
destructor
manipulated
using
acaricide
treatment.
Infestation
levels
of
V.
destructor
(mites/bee)
were
manipulated
using
acaricide
treatment
applied
at
different
moments
(July,
August,
September
or
not
treated
at
all).
For
the
expected
mite
infestation,
we
used
a
simplified
curve
from
mite
infestation
in
Figure
1,
with
an
exponential
increase
in
mite
infestation
until
October,
after
which
the
infestation
remained
equal.
Efficacy
of
the
acaricide
Thymovar
(July,
August,
September)
was
assumed
to
be
90%,
while
efficacy
of
oxalic
acid
(December)
was
assumed
to
be
95%.
doi:10.1371/journal.pone.0036285.9002
experiment
was
performed
in
two
consecutive
years
as
environ-
mental
conditions
such
as
weather
or
food
resources
are
expected
to
also
affect
winter
bee
development
and
colony
survival.
Materials
and
Methods
Experiment
The
fieldwork
took
place
in
2005/2006
and
2006/2007
at
an
apiary
of
Wageningen
UR,
The
Netherlands
(51°59'32.35"N,
5°39'46.81"E).
Colonies
(N
=
24)
were
kept
in
one-story
wooden
hives
with
10
frames
and
contained
brood
in
all
developmental
phases.
In
the
first
year
(2005/2006),
mite
fall
was
monitored
in
the
colonies
for
one
week
in
July.
The
colonies
with
the
lowest
daily
mite
fall
(2.9±0.78,
N
=
6)
were
used
to
represent
the
low
V.
destructor
infestation
from
July
onwards.
The
remaining
colonies
were
randomly
allocated
to
3
groups:
treated
in
August,
treated
in
September,
or
not
treated
at
all.
The
mean
daily
mite
fall
in
these
groups
did
not
differ
from
each
other
(overall
mean
daily
mite
fall
18.8±
3.5,
N=
18),
but
were
all
higher
compared
to
the
colonies
with
low
infestation
from
July
onwards
(daily
mite
fall
was
Logl
0-
transformed,
Anova,
F3
,
20
=
9.31,
P<0.001,
Sidak
post
hoc
test).
In
the
second
year
(2006/2007),
colonies
were
randomly
allocated
to
4
groups:
treated
in
July,
treated
in
August,
treated
in
September,
or
not
treated
at
all.
Colonies
were
treated
with
the
acaricide
Thymovar®
during
three
weeks
in
the
allocated
month.
The
experiment
in
the
second
year
was
performed
with
new
colonies.
Colonies
that
became
queenless
or
swarmed
were
removed
from
the
study.
Daily
mite
fall
in
debris
was
monitored
to
give
an
indicative
efficacy
of
the
Thymovar®
treatment
during
and
after
the
treatment
periods,
starting
in
August.
Outside
these
periods,
mite
fall
was
counted
once
a
week
(trapping
period
of
4
days
with
a
bottom
board)
to
get
an
indication
of
the
infestation
level.
In
winter
(November/December),
when
there
was
no
more
brood,
all
colonies
were
treated
with
an
oxalic
acid
solution
(trickling,
37
gr
oxalic
acid
dihydrate
in
1
L
sugar
water,
1:1
weight
ratio
for
sucrose:
water).
Mite
fall
was
counted
after
trapping
for
one
week
continuously
following
the
oxalic
acid
treatment.
Thereafter,
mite
fall
was
monitored
every
two
weeks
(trapping
period
of
2
days).
C
01
a)
in
0
z
a)
0
Not
treated
-----July
-
August
-
September
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n
July
August
September
Not
treated
b
ab
a
b
I
a
a
a
b
a
b
ab
a
ab
a
a
aa
aLI
a
a
a
Da
i
ly
m
ite
fa
ll
E
250
200
150
100
50
0
100
80
60
40
20
0
previous
month.
Sidak
posthoc
tests
were
used
for
pair
wise
comparison
of
differences
between
means.
In
2005/2006,
one
colony
from
the
group
treated
in
September
was
excluded
from
the
analysis
due
to
missing
data
on
mite
fall
for
several
months.
One
colony
(treated
in
September)
missed
data
on
mite
fall
only
in
August.
We
interpolated
this
missing
data
in
August
using
data
from
another
colony,
which
was
selected
based
on
similar
mite
fall
in
September.
Two
colonies
of
the
group
that
was
not
treated
lacked
data
in
March
and
April
due
to
mortality
of
these
colonies.
To
be
able
to
use
the
Repeated
measures
ANOVA,
we
estimated
the
mite
fall
in
these
colonies
to
be
similar
to
the
highest
mite
fall
found
in
the
months
March
and
April
for
all
treatments.
Slight
changes
in
the
estimated
mite
fall
(approx.
10%)
did
not
qualitatively
change
the
results.
Final
number
of
colonies
used
in
the
Repeated
measures
ANOVA
for
mite
fall
in
2005/2006
were
6
(treated
in
July),
7
(treated
in
August),
5
(treated
in
September),
and
5
(not
treated).
In
2006/2007,
in
total
11
colonies
were
excluded
from
the
analysis
for
mite
fall
due
to
missing
data
on
mite
fall
for
several
months:
two
colonies
in
the
group
treated
in
July,
two
in
the
group
treated
in
August,
one
in
the
group
treated
in
September,
and
eight
in
the
group
that
was
not
treated.
All
these
excluded
colonies
were
lost
between
October
and
November
2006,
possibly
due
to
high
V.
destructor
infestation.
To
test
whether
excluded
colonies
showed
higher
mite
fall
until
October
than
the
remaining
colonies,
we
used
the
Repeated
measures
ANOVA
for
mite
fall
in
August
to
October,
for
excluded
colonies
(N
=
11)
and
colonies
still
in
the
experiment
(N
=
24).
Final
number
of
experimental
colonies
used
0.60
-
0
0
0.50
-
-0
g
0.40
0.30
-
A
oJuly
August
September
Not
treated
a
0
b
b
c
c`
1
0
)
0.00
S
0.15
-
0.06
-
to
0.03
-
a)
on
a)
0.00
")\
B
I
I
a
a
a
a
a
b
Figure
4.
Bee
survival
as
a
function
of
time
and
treatment
in
2005/2006
(A)
and
2006/2007
(B).
Bee
survival
(fraction
d100)
was
the
predicted
fraction
of
bees
that
was
still
alive
at
the
age
of
100
days,
and
calculated
using
a
Cox
Proportional
Hazards
Model.
Time
was
the
marking
date
of
the
cohort
(scatterplot).
Different
months
of
acaricide
application
show
the
treatments,
where
letters
denote
significant
differences
(over
all
marking
dates).
doi:10.1371/journal.pone.0036285.9004
v
d
2
3
t
a:3
s
ec
s
ec
06-
peg
odr.-
,
4
0•
4
4;1'
•".)--
..>"
19"
.
ti
Z
Marking
of
cohort
Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
Counting
mite
fall
has
been
shown
to
be
effective
to
estimate
the
population
of
mites
[15,16].
In
half
of
the
colonies
of
each
experimental
group,
the
number
of
capped
brood
cells
was
estimated
by
superimposing
a
grid
with
2.5
x2.5
cm
squares
over
the
brood
area.
Solid
squares
were
counted
directly
and
partial
squares
estimated.
The
number
of
brood
cells
was
then
calculated
from
the
number
of
grids
multiplied
by
25
brood
cells
(we
counted
400
cells
in
one
dm
2
).
Brood
was
measured
every
two
weeks
from
mid-August
until
mid-
November.
During
2006/2007,
due
to
the
high
winter
temper-
atures,
brood
measurements
were
continued
every
month
until
mid-April.
Every
fortnight,
cohorts
of
approximately
100
newly
emerged
bees
were
marked
with
a
unique
colour
(colour
marker
Posca)
and
returned
to
their
original
colony.
In
2005
marking
cohorts
started
in
July,
resulting
in
eight
cohorts
in
four
(out
of
six)
colonies
per
treatment.
In
2006
marking
started
in
August,
resulting
in
seven
cohorts
in
four
(out
of
six)
colonies
per
treatment.
Marking
cohorts
was
stopped
at
the
beginning
of
November
in
both
years.
At
equal
intervals,
the
presence
of
bees
from
previously
marked
cohorts
was
recorded.
Based
on
the
unique
colour
the
age
of
the
bees
could
be
determined.
Recording
cohort
survival
continued
until
mid-April
the
following
year
or
until
no
more
marked
bees
were
observed.
If
colonies
could
not
be
examined
during
winter,
it
was
assumed
that
worker
mortality
was
constant.
After
winter
in
April,
the
size
of
the
colony
was
estimated
by
counting
the
number
of
frames
with
bees.
Non-surviving
colonies
had
zero
frames
with
bees.
Statistics
To
test
whether
the
weather
differed
between
the
two
years,
the
differences
in
ambient
temperature
were
tested
with
a
paired
t-test
(paired
for
month)
for
the
period
July—November
and
the
period
December—April
separately
in
2005/2006
and
2006/2007.
Mean
daily
mite
fall
per
colony
was
calculated
per
month.
Repeated
measures
ANOVAs
were
used
to
test
mite
fall
for
2005/
2006
and
2006/2007
separately,
as
mean
daily
mite
fall
in
one
month
was
assumed
to
be
correlated
to
mean
daily
mite
fall
in
the
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
Figure
3.
Mean
daily
mite
fall
in
2005/2006
(A)
and
2006/2007
(B).
Colonies
were
treated
with
Thymovar®
in
July
(white
bars),
August
(grey
bars),
September
(dark
grey
bars),
or
not
treated
at
all
(black
bars).
All
colonies
were
treated
in
December
using
oxalic
acid
(3%).
Letters
denote
significant
differences
between
treatments
within
each
month.
No
letters
mean
no
significant
differences
between
treatments
were
found.
Differences
between
months
were
not
given.
doi:10.1371/journal.pone.0036285.9003
PLoS
ONE
I
www.plosone.org
3
April
2012
I
Volume
7
I
Issue
4
I
e36285
Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
in
the
Repeated
measures
ANOVA
for
mite
fall
in
2006/2007
was
7
(treated
in
July),
6
(August),
6
(September),
and
5
(not
treated).
We
calculated
the
survival
rate
for
each
cohort
of
bees
marked
in
a
colony,
using
survival
analysis
with
Cox
Proportional
Hazards
Models
for
treatment
(timing
of
acaricide
application),
for
the
number
of
days
since
the
cohorts
were
marked
(is
equal
to
the
day
the
bees
in
that
cohort
were
born),
and
for
2005/2006
and
2006/
2007
separately.
In
addition
to
testing
the
differences
between
treatments
and
the
differences
over
time,
the
Cox
Proportional
Hazards
Model
was
used
to
predict
the
fraction
of
bees
in
a
cohort
that
is
still
alive
(in
statistical
terms
this
is
called
the
predicted
survival
probability)
at
a
certain
age
of
the
cohort,
from
here
onwards
called
'bee
survival'.
To
compare
treatments
over
time,
we
calculated
bee
survival
at
100
days
(fraction
of
bees
still
alive
at
the
age
of
100
days).
As
summer
bees
only
live
for
about
35
days,
while
winter
bees
live
for
about
135
days
[12]
or
150
days
[17],
we
assume
that
bees
that
are
alive
after
100
days
are
winter
bees.
Low
bee
survival
at
100
days
means
that
the
mean
lifespan
of
the
bees
in
the
cohort
is
short.
Consequently
this
means
for
winter
bees
that
fewer
bees
will
survive
until
spring
and
be
able
to
contribute
to
spring
development
of
the
colony.
To
test
whether
bee
survival
at
100
days
differed
between
2005/2006
and
2006/2007,
Repeated
measures
ANOVA
was
used.
As
the
days
the
cohorts
were
marked
did
not
coincide
perfectly
between
the
years,
we
paired
the
days
most
similar
for
both
years
(maximum
difference
was
2
days)
and
excluded
the
cohorts
marked
in
July
2005
(no
cohorts
were
marked
in
July
2006).
Additionally,
as
we
had
only
one
mean
value
per
day
of
marking
per
treatment,
the
treatments
were
pooled
(N
=
4
per
day
of
marking).
Repeated
measures
ANOVAs
were
used
to
analyse
the
change
in
the
number
of
capped
brood
cells
over
time
in
2005/2006
and
2006/2007
separately.
When
colonies
were
lost
during
the
experiment,
brood
measurements
of
other
colonies
within
the
experiment
were
used
to
continue
the
brood
measurements.
In
2005/2006,
capped
brood
cells
were
counted
from
August
to
November
and
in
April.
Between
November
2005
and
April
2006,
actual
counts
of
capped
brood
cells
were
suspended
due
to
cold
temperatures
and
dense
clustering
of
bees.
The
final
number
of
colonies
for
counting
capped
brood
cells
was
3
for
colonies
treated
in
July,
3
for
colonies
treated
in
August,
2
for
colonies
treated
in
September,
and
3
for
colonies
that
were
not
treated.
In
2006/
2007,
brood
cells
were
counted
continuously
from
August
to
April.
The
final
number
of
colonies
for
counting
capped
brood
cells
was
4
for
colonies
treated
in
July,
3
for
colonies
treated
in
August,
3
for
colonies
treated
in
September,
and
3
for
colonies
that
were
not
treated.
To
test
whether
the
fraction
of
winter
bees
in
a
cohort
increased
during
the
decrease
of
brood
in
autumn
we
used
a
General
Linear
Model.
Mean
bee
survival
at
100
days
of
the
different
treatments
(timing
of
acaricide
application,
fixed
factor)
was
tested
as
a
function
of
the
number
of
brood
cells
(as
covariate)
for
2005/2006
and
2006/2007
separately.
Possible
interactions
between
treat-
10000
in
a)
L.)
15
4000
O
0
co
2000
a
A
b
July
August
September
Not
treated
8000
6000
d
e
0
10000
-
a
8000
-
a
cd
U)
4_
6000
-
O
-
0
4000
-
O
O
05
2000
-
0
a
b
r
bd
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
Figure
5.
Mean
number
of
capped
brood
cells
in
2005/2006
(A)
and
2006/2007
(B).
Colonies
were
treated
with
Thymovar®
in
July
(white
bars),
August
(grey
bars),
September
(dark
grey
bars),
or
not
treated
at
all
(black
bars).
Number
of
capped
brood
cells
between
December
2005
and
March
2006
were
not
measured
due
to
cold
winter
temperatures.
Letters
show
significant
differences
between
months.
doi:10.1371/journal.pone.0036285.9005
PLoS
ONE
I
www.plosone.org
4
April
2012
I
Volume
7
I
Issue
4
I
e36285
Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
Bee
Su
rv
iv
a
l
(
fr
ac
tion
d
io0
)
ments
in
relation
to
the
decrease
in
brood
were
added
to
show
differences
in
the
rate
of
change
in
bee
survival.
Sidak
posthoc
tests
for
pair
wise
comparison
were
used
to
test
for
differences
between
treatments.
To
test
the
differences
in
colony
size
in
April
between
the
treatments,
we
calculated
the
mean
fraction
of
frames
that
was
occupied
with
bees
in
April
using
a
Generalized
Linear
Model.
If
a
colony
had
died
before
April,
the
number
of
frames
occupied
by
bees
was
zero.
The
mean
fraction
of
frames
was
estimated
with
the
number
of
occupied
frames
in
April
as
dependent
variable
and
the
10
frames
that
were
available
in
each
hive
as
fixed
number
of
trials
(binomial
distribution
and
logit
link
function).
Sidak
posthoc
tests
for
pair
wise
comparison
were
used
to
test
differences
in
the
mean
fraction
of
frames
between
treatments.
A
Pearson
correlation
was
used
to
test
if
there
was
a
correlation
between
bee
survival
at
100
days
for
the
cohort
that
was
marked
(born)
in
November
and
the
fraction
of
frames
occupied
with
bees
in
April.
Results
Ambient
temperature
The
mean
ambient
temperature
during
summer
and
autumn
(July—November)
in
The
Netherlands
did
not
differ
between
2005
(14.0±1.9°C)
and
2006
(15.9±2.2°C;
paired
t-test:
t
4
=
—2.38,
P
=
0.08).
Mean
temperature
between
December
2005
to
April
2006
was
however
lower
(4.3±1.3°C)
than
between
December
2006
and
April
2007
(8.1±1.3°C;
paired
t-test:
t
4
=
—7.35,
P
<
0.0
I).
Acaricide
treatment
effectiveness
(mite
fall)
In
2005/2006,
mean
daily
mite
fall
differed
between
the
treatments
per
month
(Repeated
measures
ANOVA:
treatment
F
3
,
19
=
2.76,
P
=
0.07;
month
F
8
,
152
=
32.87,
P<0.001;
treatment
x
month
F
24
,
152
=
2,39,
P
=
0.001;
Figure
3A).
As
can
be
expected,
mean
daily
mite
fall
in
August
was
highest
for
colonies
treated
in
August,
and
highest
in
September
for
colonies
treated
in
September.
In
2006/2007,
mean
daily
mite
fall
also
differed
between
the
treatments
per
month
(Repeated
measures
ANOVA:
treatment
F
3
,
20
=
7.63,
P
=
0.001;
month
F
8
,
160
=
41.38,
P<0.001;
treatment
xmonth
F
24
,
152
=
8.17,
P<0.001;
Figure
3B).
Again,
daily
mite
fall
in
August
was
highest
for
colonies
treated
in
August,
and
highest
in
September
for
colonies
treated
in
September.
Daily
mite
fall
for
colonies
that
were
not
treated
remained
high
during
the
year.
In
2006,
colonies
that
were
lost
between
October
and
November
and
excluded
from
the
analysis
above
indeed
showed
higher
daily
mite
fall
(overall
38.7±4.9)
than
colonies
included
in
the
analysis
(overall
18.6±3.3),
where
the
daily
mite
fall
increased
with
time
(month),
but
more
for
the
colonies
excluded
than
for
colonies
included
in
the
analysis
(Repeated
measures
ANOVA:
in/
excluded
F
1
,
33
=
11.33,
P
=
0.002;
month
F
1
,
33
=
13.82,
P
=
0.001;
in/excluded
xmonth
F
1
,
33
=
5.59,
P
=
0.02).
Bee
survival
Mean
survivorship
curves
for
marked
cohorts
of
bees
are
shown
in
Figure
SI.
For
the
survival
analysis,
the
Cox
Proportional
Hazards
Models
used
6398
uncensored
cases
and
353
censored
cases
for
2005/2006,
and
8458
uncensored
cases
and
600
censored
cases
for
2006/2007.
The
cumulative
survival
curves
for
the
different
treatments
over
time
clearly
showed
a
lower
bee
survival
in
colonies
that
were
not
treated
compared
to
all
other
treatments
in
both
2005/2006
(Wald
=
123.2,
df
=
3,
P<0.001)
and
2006/2007
(Wald
=
87.2,
df=
3,
P<0.001;
Figure
S2).
We
found
that
the
cumulative
survival
increased
with
time
in
both
2005/2006
(Wald=
435.1,
df=
6,
P<0.001)
and
2006/2007
(Wald
=
200.4,
df=
6,
P<0.001;
Figure
S2),
suggesting
an
increasing
fraction
of
winter
bees
in
the
cohorts.
Bee
survival
at
day
100
(fraction
of
bees
still
alive
at
the
age
of
100
days)
was
predicted
by
the
model
as
a
function
of
time
and
treatment
in
2005/2006
and
2006/2007
(Figure
4).
For
both
2005/2006
and
2006/2007,
using
day
50,
75
or
120
did
not
qualitatively
change
the
results.
When
marked
in
2005,
bee
survival
at
100
days
was
higher
than
in
2006
from
August
24
th
onwards
(2006,
coinciding
with
August
25
th
for
2005),
and
this
difference
became
larger
towards
the
end
(Repeated
measures
ANOVA:
year
F
1
,
21
=
805.70,
P<0.001;
marking
day
F
6
,
21
=
13.83,
P<0.001;
year
xmarking
day
F6,21
=
29.31,
P<0.001).
For
example,
from
bees
that
emerged
on
November
4
th
2005
44±3%
was
still
alive
at
an
age
of
100
days,
while
from
bees
that
emerged
on
November
2'
1
2006
only
14
-
±
1
%
was
still
alive
at
an
age
of
100
days.
Colony
development
(brood)
In
2005/2006,
the
number
of
capped
brood
cells
decreased
between
August
and
November
(Repeated
measures
ANOVA:
treatment
F
3
,
7
=
1.59,
P
=
0.28;
month
F
4
,
28
=
223.65,
P<0.001;
treatment
xmonth
F
12
,
28
=
1.20,
P
=
0.33;
Figure
5A).
Brood
rearing
had
not
yet
shown
the
expected
spring
increase
in
April
2006
for
any
of
the
treatments.
In
2006/2007,
the
number
of
capped
brood
cells
also
decreased
between
August
and
November
(Repeated
measures
ANOVA:
treatment
F
3
,
9
=
3.89,
P
=
0.05;
month
F
9
,
72
=
38.25,
P
<
O.
001
;
treatment
xmonth
F
24
,
72
=
1.20,
P
=
0.38;
Figure
5B).
Brood
rearing
continued
at
a
low
rate
during
winter
and
was
much
increased
in
April
2007
for
all
treatments.
Although
the
Repeated
measures
ANOVA
showed
a
borderline
significant
effect
of
treatment
for
2006/2007,
the
Sidak
posthoc
test
did
not
show
differences
between
treatments
(the
number
of
A
0.45
-
8
A_
O.
0.30
-
••
0.15
0
0.00
0.20
0
c
0.15
0
ea
0.10
n
0.05
CO
0.00
0
2000
4000
6000
8000
10000
Brood
(#
of
cells)
Figure
6.
Bee
survival
as
a
function
of
brood
in
2005/2006
(A)
and
2006/2007
(B).
Bee
survival
(fraction
d100),
the
predicted
fraction
of
bees
that
was
still
alive
at
the
age
of
100
days,
as
function
of
the
number
of
capped
brood
cells
for
the
different
months
of
acaricide
application.
Symbols
show
means
per
marking
day.
doi:10.1371/journal.pone.0036285.9006
0.60
-
0July
August
September
Not
treated
El
0
B
OA.
IL
%
0
NOA
A
••
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5
April
2012
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Volume
7
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4
I
e36285
Occ.
Fr
ames
(
fr
ac
tion
Ap
r
il
)
Occ.
Fr
ames
(
fr
ac
tion
Ap
r
;,
)
b
b
Jul
Aug
Sep
NT
a
b
0.4
LL
0.2
-
6
0
0.0
.1u1
Aug
Sep
NT
Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
capped
brood
cells
for
colonies
treated
in
July
was
almost
higher
than
brood
for
colonies
treated
in
August,
Sidak
P
=
0.08).
Bee
survival
in
relation
to
number
of
capped
brood
cells
In
2005/2006,
bee
survival
increased
with
a
decrease
in
number
of
capped
brood
cells
(General
Linear
Model:
treatment
F
3
,
23
=
6.52,
P<0.01;
brood
F
1
,
23
=
162.39,
P<0.001;
Figure
6A;
if
the
interaction
was
included,
then
both
the
interaction
between
treatment
xbrood
and
the
main
effect
treatment
were
not
significant).
In
relation
to
brood,
there
was
a
lower
bee
survival
for
colonies
that
were
not
treated
than
for
colonies
treated
in
July
or
September,
but
not
lower
than
colonies
treated
in
August.
In
2006/2007,
bee
survival
also
increased
with
a
decrease
in
number
of
capped
brood
cells
(General
Linear
Model:
treatment
F
3
,
23
=
3.60,
P<0.05;
brood
F
1
,
23
=
38.59,
P<0.001;
Figure
6B;
if
the
interaction
was
included,
then
both
the
interaction
between
treatment
xbrood
and
the
main
effect
treatment
were
not
significant).
There
was
a
lower
bee
survival
for
colonies
that
were
not
treated
than
for
colonies
treated
in
July,
but
not
compared
to
colonies
treated
in
August
or
September.
Winter
survival
Between
November
2005
and
April
2006,
four
colonies
were
lost
in
the
group
not
treated
with
acaricide,
while
no
winter
colony
loss
occurred
in
the
other
groups.
The
fraction
of
frames
(out
of
10)
that
were
occupied
with
bees
in
April
2006
was
the
lowest
for
colonies
that
were
not
treated
in
2005
(Generalized
Linear
Model:
Wald
Chi-Square
=
38.1,
df=
3,
P<0.001;
Figure
7A
insert),
and
increased
with
an
increase
in
bee
survival
(Pearson
correlation:
r
=
0.98,
n
=
4,
P
=
0.02;
Figure
7A).
During
the
winter
of
2006/
2007,
no
colonies
were
lost.
The
fraction
of
frames
(out
of
10)
that
was
occupied
with
bees
in
April
2007
was
highest
for
the
colonies
that
were
treated
with
acaricide
in
July
2006
(Generalized
Linear
Model:
Wald
Chi-Square
=
9.2,
df=
3,
P
=
0.027;
Figure
7B
insert),
but
did
not
relate
to
bee
survival
(Pearson
correlation:
r
=
0.42,
n
=
4,
P
=
0.58;
Figure
7B).
For
the
relation
between
the
fraction
of
frames
that
were
occupied
with
bees
in
April
and
bee
survival,
data
from
November
was
used
as
an
example:
the
relationships
were
similar
for
all
days
the
cohorts
were
marked,
the
trend
only
showed
lower
bee
survival
for
cohorts
marked
earlier.
Discussion
In
this
study,
we
found
that
low
V.
destructor
infestation
levels
during
the
development
of
winter
bees
resulted
in
an
increase
in
lifespan
of
bees
compared
to
colonies
that
were
not
treated
and
that
had
higher
infestation
levels.
Acaricide
treatment
before
the
expected
transition
period
from
summer
to
winter
bees
resulted
in
1.0
A
0.8
0.6
0.4
0.
2
0.0
0.3
1.0
B
0.8
0.6
0.4
0.2
0.0
0.0
+
1.0
f
.
0.5
0.6
I
0.4
u-
0.2
0
0.0
0.4
0.5
0.6
0.1
0.2
0.3
Bee
Survival
(fraction
dloo
)
Figure
7.
Winter
survival
as
a
function
of
bee
survival
in
November
2005
(B)
and
2006
(B).
Fraction
of
frames
occupied
with
bees
in
a
colony
in
April
in
relation
to
bee
survival
at
100
days
for
the
cohorts
marked
in
November
2005
(A)
and
November
2006
(B).
We
used
the
data
for
November
as
an
example,
the
relationship
is
similar
for
all
days
of
marking,
the
trend
only
showed
lower
bee
survival
for
cohorts
marked
earlier.
Inserts
show
the
differences
in
the
fraction
of
frames
occupied
between
for
the
different
treatments
(timing
of
acaricide
application,
NT=
not
treated).
Letters
indicate
significant
differences.
doi:10.1371/journal.pone.0036285.9007
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Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
the
highest
lifespan
of
bees.
Colonies
with
low
infestation
levels
had
fewer
losses
in
number
of
bees
and
higher
survival
during
and
after
winter.
A
large
number
of
bees
in
the
bee
colony
at
the
start
of
the
growing
season
in
temperate
regions
has
indeed
shown
to
increase
survival
and
production
of
bee
colonies
[18].
Several
studies
reported
the
decrease
in
lifespan
of
individual
bees
due
to
V.
destructor
infestation
[6,7,19]
or
the
altered
physiology
in
bees
suggesting
a
decrease
in
lifespan
[8].
Here,
we
link
the
decreased
lifespan
of
individual
bees
due
to
V.
destructor
infestation
to
colonies
losses
in
at
least
some
circumstances.
Mattila
et
al.
[12]
showed
an
increase
in
bee
longevity
between
August
and
the
beginning
of
November,
which
fully
agrees
with
our
findings:
the
number
of
capped
brood
cells
decreased
drastically
between
August
and
November,
while
at
the
same
time,
the
lifespan
of
the
bees
increased
indicating
the
transition
of
short-lived
to
long-lived
winter
populations
[20].
When
low
infestation
of
V.
destructor
occurred
earlier
in
the
period
of
winter
bee
transition,
lifespan
of
the
bees
increased
and
consequently
the
winter
survival
of
the
colonies
increased,
which
supports
previous
findings
by
Delaplane
and
Hood
[13]
and
Currie
and
Gatien
[14].
In
our
study,
however,
mean
lifespan
(estimated
by
bee
survival
at
100
days)
was
longer
during
the
winter
in
2005/2006,
compared
to
the
winter
of
2006/2007.
A
variety
of
stress-related
factors
such
as
winter
temperatures
or
foraging
conditions
in
autumn,
could
have
contributed
to
the
variation
in
lifespan
between
years.
The
much
shorter
lifespan
for
bees
during
the
winter
2006/2007
at
least
suggests
that
bees
were
more
active
during
this
winter.
Possibly
due
to
the
observed
rearing
of
brood,
as
long
lifespan
is
inhibited
by
brood
pheromones
[21]
and
reduced
by
brood
rearing
activities
depleting
body
reserves
[20,22].
This
shorter
lifespan,
however,
may
have
been
less
problematic
due
to
the
earlier
start
of
spring
[8]
illustrated
by
the
high
number
of
brood
cells
in
April
2007
compared
to
the
year
before.
Although
winter
temperature
was
not
included
as
a
replicated
treatment,
we
observed
that
mean
lifespan
(estimated
by
bee
survival
at
100
days)
was
longer
during
the
colder
winter
in
2005/
2006,
compared
to
the
relatively
mild
winter
of
2006/2007.
Mean
longevity
in
the
study
of
Mattila
et
al.
[12]
was
longer
than
in
our
study,
for
comparison:
on
October
6
th
longevity
ranged
between
125-150
days
in
the
study
of
Mattila
et
al.
[12],
while
in
our
study
on
this
date
mean
longevity
was
62
days
for
2006/2007
and
93
for
2005/2006
(calculated
using
the
method
described
in
Matilla
et
al.
[12]).
Mattila
et
al.
[12]
performed
their
experiments
in
the
south
of
Manitoba,
Canada,
which
has
approximately
the
same
latitude
as
The
Netherlands,
but
has
a
continental
climate
characterized
by
large
annual
amplitudes
in
temperature
instead
of
an
oceanic
climate
as
in
our
site
with
narrow
annual
temperature
amplitudes.
The
even
lower
winter
temperatures
in
Canada
compared
to
the
Netherlands
can
maybe
explain
the
longer
lifespan
of
the
Canadian
bees.
We
therefore
hypothesize
that
the
negative
effect
of
V.
destructor
(i.e.,
shortened
lifespan
of
winter
bees
and
possible
colony
loss)
is
larger
under
colder
winter
conditions.
Colony
survival,
measured
by
the
number
of
frames
with
bees
occupied
in
April,
was
highest
with
treatment
against
V.
destructor
applied
in
July,
due
to
the
longest
lifespan
of
the
bees
(bee
survival
at
100
days)
in
autumn
for
these
colonies.
Delaplane
and
Hood
[13]
also
studied
the
effects
of
timing
of
acaricide
treatment
(with
Apistan)
on
honeybee
colonies
parasitized
by
V.
destructor,
where
type
of
one-story
hives,
colony
sizes
and
amounts
of
brood
were
comparable
to
our
experiment.
They
found
that
colony
survival
and
colony
size,
measured
in
December,
was
higher
by
acaricide
treatment
in
August
(in
contrast
to
treatment
in
June
or
October).
In
their
study,
colonies
treated
in
October
resulted
in
unacceptably
high
bee
mortality
in
December.
Mite
fall
before
treatment
of
these
colonies
was
145±30
mites
per
18
±5
h,
which
was
much
higher
than
mite
fall
in
November
in
our
study
(max.
32
-
±
11
mites
per
24
h;
mite
fall
in
December
in
our
study
was
not
representative
for
'natural'
mite
fall
due
to
the
acaricide
treatment
in
this
month).
In
our
study,
however,
at
this
relatively
low
level
of
mite
fall,
colony
loss
already
occurred.
Our
late
treatment
(September)
did not
show
an
increase
in
colony
size
(in
April),
and
nor
did
theirs
(October,
resulted
in
a
45%
decline
in
colony
size
in
December).
Acaricide
treatments
to
kill
V.
destructor
in
late
autumn
may
thus
fail
to
prevent
losses
of
colonies
because
many
of
the
adult
bees
are
no
longer
able
to
survive
until
spring
[8].
We
manipulated
the
level
of
V.
destructor
infestation
by
using
acaricide
treatments
at
different
moments.
This
acaricide
treatment
with
Thymovar®
was
effective
because
mitefall
was
indeed
increased
during
the
month
the
acaricide
treatment
was
applied.
The
pattern
of
mite
fall
directly
after
the
acaricide
treatment
for
the
different
moments
(Figure
3)
confirms
with
the
expected
infestation
level
after
the
month
of
treatment
(Figure
2).
The
efficacy
of
Thymovar®
as
an
acaricide
has
been
shown
before:
72%
for
one-story
and
94%
for
two-story
colonies
[23],
or
97%
for
one-story
colonies
with
low
amount
of
brood
[24].
Although
mite
fall
was
reduced
after
the
acaricide
treatment
in
July,
August
or
September,
it
was
not
as
much
reduced
as
after
the
treatment
using
oxalic
acid
in
December.
Oxalic
acid
however
only
affects
mites
in
the
phoretic
phase,
which
is
the
predominant
phase
during
winter
when
brood
rearing
has
stopped
or
is
reduced
[25,26].
This
is
supported
by
the
slightly
higher
mite
fall
during
winter
2006/2007
compared
to
2005/2006,
and
the
most
likely
higher
amount
of
reared
brood
(assumed
during
winter
2005/
2006,
not
measured).
Previous
studies
showed
that
V.
destructor
infestation
reduces
the
body
weight
and
protein
content
of
individual
bees,
which
shortens
their
lifespan
[6-8].
Our
study
supports
these
findings
and
shows
the
relation
between
decreased
lifespan
of
individual
bees
and
increased
colony
losses.
Additionally,
colonies
treated
earlier
in
the
season
had
reduced
V.
destructor
infestation
before
the
development
of
winter
bees
resulting
in
longer
bee
lifespan
and
higher
colony
survival
after
winter
(Figure
7).
This
study
contributes
to
theory
about
the
multiple
causes
for
the
recent
elevated
colony
losses
in
honey
bees.
Our
study
shows
that
high
V.
destructor
infestation
during
the
transition
to
winter
bees
can
cause
these
colonies
losses
due
to
decreased
lifespan
of
winter
bees.
We
can
expect
that
other
environmental
stresses,
such
as
pesticides,
other
pathogens,
decreased
food
availability,
or
reduced
diversity
of
this
food
[1,3,27],
in
combination
with
V.
destructor
will
further
reduce
lifespan
of
bees
and
increase
colony
losses
during
and
after
winter.
Supporting
Information
Figure
Si
Mean
survivorship
curves
for
cohorts
of
bees
marked
in
2005/2006
(left)
and
2006/2007
(right).
Cohorts
of
bees
were
marked
at
14-day
intervals
for
each
acaricide
treatment:
July
(open
diamonds),
August
(grey
squares),
September
(dark
grey
triangles),
and
not
treated
at
all
(black
circles).
Each
line
shows
the
mean
survival
of
1
to
4
cohorts.
During
the
winter
of
2005/2006,
actual
counts
of
marked
bees
were
suspended
due
to
cold
temperatures;
mortality
was
assumed
to
be
constant
for
that
period.
(TIF)
Figure
S2
Cumulative
survival
curves
for
2005/2006
(top)
and
2006/2007
(bottom),
per
acaricide
treatment
(left),
and
for
the
marking
date
of
the
cohorts
(right).
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ONE
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7
April
2012
I
Volume
7
I
Issue
4
I
e36285
Varroa
Mite
Decreases
Honey
Bee
Winter
Survival
Cumulative
survival
curves
were
calculated
from
the
Cox
Proportional
Hazards
Models
for
cohorts
of
bees
marked.
For
the
survival
analysis,
we
had
6398
uncensored
cases
and
346
censored
cases
for
2005/2006,
and
8458
uncensored
cases
and
547
censored
cases
for
2006/2007.
During
the
winter
of
2005/
2006,
actual
counts
of
marked
bees
were
suspended
due
to
cold
temperatures;
mortality
was
assumed
to
be
constant
for
that
period.
(TIF)
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1.
Ellis
JD,
Evans
JD,
Pettis
J
(2010)
Colony
losses,
managed
colony
population
decline,
and
Colony
Collapse
Disorder
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the
United
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Apic
Res
49:
134-136.
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Conte
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Ellis
M,
Ritter
W
(2010)
Varroa
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bee
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of
the
colony
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(2010)
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al.
(2010b)
Declines
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Jong
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de,
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Acknowledgments
Jeroen
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Daan
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Christ
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Contributions
Conceived
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LG
BC
11JMvdS
TB.
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LG
BCIUMvdS
TB.
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8
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I
Volume
7
I
Issue
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e36285