Toxicity of Selected Acaricides to Honey Bees (Apis mellifera) and Varroa (Varroa destructor Anderson and Trueman) and Their Use in Controlling Varroa within Honey Bee Colonies


Gregorc, Aš.; Alburaki, M.; Sampson, B.; Knight, P.R.; Adamczyk, J.

Insects 9(2): 1-16

2018


The efficacies of various acaricides in order to control a parasitic mite, the Varroa mite, <i>Varroa destructor</i>, of honey bees, were measured in two different settings, namely, in laboratory caged honey bees and in queen-right honey bee colonies. The Varroa infestation levels before, during, and after the acaricide treatments were determined in two ways, namely: (1) using the sugar shake protocol to count mites on bees and (2) directly counting the dead mites on the hive bottom inserts. The acaricides that were evaluated were coumaphos, tau-fluvalinate, amitraz, thymol, and natural plant compounds (hop acids), which were the active ingredients. The acaricide efficacies in the colonies were evaluated in conjunction with the final coumaphos applications. All of the tested acaricides significantly increased the overall Varroa mortality in the laboratory experiment. Their highest efficiencies were recorded at 6 h post-treatment, except for coumaphos and thymol, which exhibited longer and more consistent activity. In the honey bee colonies, a higher Varroa mortality was recorded in all of the treatments, compared with the natural Varroa mortality during the pretreatment period. The acaricide toxicity to the Varroa mites was consistent in both the caged adult honey bees and workers in the queen-right colonies, although, two of these acaricides, coumaphos at the highest doses and hop acids, were comparatively more toxic to the worker bees.

It
insects
Article
Toxicity
of
Selected
Acaricides
to
Honey
Bees
(Apis
mellifera)
and
Varroa
(Varroa
destructor
Anderson
and
Trueman)
and
Their
Use
in
Controlling
Varroa
within
Honey
Bee
Colonies
Ala
Gregorc
1
'
2
'
4
,
Mohamed
Alburaki
3
,
Blair
Sampson
4
,
Patricia
R.
Knight
1
and
John
Adamczyk
4
1
Center
for
Costal
Horticulture
Research,
Mississippi
State
University,
Poplarville,
MS
39470,
USA;
prk3@msstate.edu
2
Agricultural
Institute
of
Slovenia,
1000
Ljubljana,
Slovenia
and
Faculty
of
Agriculture
and
Life
Sciences,
University
of
Maribor,
2000
Maribor,
Slovenia
3
Department
of
Biological
Sciences,
The
University
of
Southern
Mississippi,
Hattiesburg,
MS
39406,
USA;
Mohamed.Alburaki@usm.edu
4
USDA
ARS,
Thad
Cochran
Southern
Horticultural
Research
Laboratory,
Poplarville,
MS
39470,
USA;
Blair.Sampson@ARS.USDA.GOV
(B.S.);
John.Adamczyk@ARS.USDA.GOV
(J.A.)
Correspondence:
ales.gregorc@kis.si
check
for
Received:
7
February
2018;
Accepted:
9
May
2018;
Published:
10
May
2018
updates
Abstract:
The
efficacies
of
various
acaricides
in
order
to
control
a
parasitic
mite,
the
Varroa
mite,
Varroa
destructor,
of
honey
bees,
were
measured
in
two
different
settings,
namely,
in
laboratory
caged
honey
bees
and
in
queen-right
honey
bee
colonies.
The
Varroa
infestation
levels
before,
during,
and
after
the
acaricide
treatments
were
determined
in
two
ways,
namely:
(1)
using
the
sugar
shake
protocol
to
count
mites
on
bees
and
(2)
directly
counting
the
dead
mites
on
the
hive
bottom
inserts.
The
acaricides
that
were
evaluated
were
coumaphos,
tau-fluvalinate,
amitraz,
thymol,
and
natural
plant
compounds
(hop
acids),
which
were
the
active
ingredients.
The
acaricide
efficacies
in
the
colonies
were
evaluated
in
conjunction
with
the
final
coumaphos
applications.
All
of
the
tested
acaricides
significantly
increased
the
overall
Varroa
mortality
in
the
laboratory
experiment.
Their
highest
efficiencies
were
recorded
at
6
h
post-treatment,
except
for
coumaphos
and
thymol,
which
exhibited
longer
and
more
consistent
activity.
In
the
honey
bee
colonies,
a
higher
Varroa
mortality
was
recorded
in
all
of
the
treatments,
compared
with
the
natural
Varroa
mortality
during
the
pretreatment
period.
The
acaricide
toxicity
to
the
Varroa
mites
was
consistent
in
both
the
caged
adult
honey
bees
and
workers
in
the
queen-right
colonies,
although,
two
of
these
acaricides,
coumaphos
at
the
highest
doses
and
hop
acids,
were
comparatively more
toxic
to
the
worker
bees.
Keywords:
Varroa
destructor;
honey
bee;
caged-bees;
varroacides
1.
Introduction
Varroa
destructor
[1]
is
a
worldwide
parasite
of
Apis,
which
causes
significant
brood
and
adult
mortality
in
colonies
of
European
honey
bees
(Apis
mellifera
L.).
Reliable
Varroa
population
diagnosis,
monitoring,
and
control
are
the
prominent
issues
in
modern
beekeeping,
which,
if
improved,
could
reduce
or
reverse
the
global
losses
of
honey
bees
and
their
colonies
[2].
If
the
Varroa
mites
are
left
untreated,
the
commercial
colonies
will
normally
die
within
three
to
five
years.
Worsening
the
situation
are
the
populations
of
the
Varroa
mites
that
have
evolved
a
resistance
to
many
of
the
synthetic
acaricides
[3-5].
Therefore,
beekeepers
increasingly
rely
on
acaricides
with
different
modes
of
action,
many
of
which
contain
essential oils
and
organic
acids
as
active
ingredients
[
].
Insects
2018,
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55;
doi:10.3390/irisects9020055
www.mdpi.com/journal/insects
Insects
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2
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A
wide
array
of
chemotherapeutic,
api-technical,
biological,
and
behavioral
methods are
available
for
the
control
of
the
Varroa
mites
within
honey
bee
colonies
[2].
To
minimize
the
pesticide
residues
in
the
colonies
and
hive
products,
and
to
thwart
the
acaricide
resistance
[8,9],
beekeepers
increasingly
rely
on
organic
oils
and
acids,
which
are
variably
effective
controls
of
Varroa.
Many
beekeepers
prefer
to
apply
synthetic
pyrethroids,
such
as
tau-fluvalinate
(Apistan
®
)
and
flumethrin
(Bayvarol
®
),
as
well
as
the
organophosphate,
coumaphos
(CheckMite
®
),
and
the
octopamine
analog,
amitraz
(Apivar
®
),
because
of
their
low
cost,
ease
of
application,
prolonged
activity,
and
perceived
high
efficacy
[10].
However,
an
over-reliance
on
these
products,
without
adequate
rotation,
is
associated
with
increasing
incidences
of
Varroa
resistance,
particularly
to
the
pyrethroids
[11-15].
Botanical-based
acaricides
are
also
commercially
registered
for
Varroa
control.
The
first
of
these
is
Apiguard
®
(Vita
Europe
Ltd.;
Basingstoke,
UK),
which
contains
the
volatile
monoterpenoid,
thymol,
an
active
ingredient
that
is
gradually
released
within
bee
colonies
from
food-grade
gel
packets
[16-18].
Experimentally,
Apiguard
®
is
shown
to
induce
76%
to
95%
of
the
mortality
in
Varroa
mites
[4,19].
In
the
fully
developed
colonies
containing
a
reproductive
queen,
workers
capped
brood,
and
honey
stores
in
combs
that
are
maintained
in
a
continental
climate,
Apiguard
®
kills
less
than
50%
of
the
mites
[
].
HopGuard
®
,
is
another
botanical-based
acaricide
with
beta
hop
acids
as
active
ingredients.
HopGuard
is
effective
against
Varroa
within
colonies,
whether
the
frames
contain
the
open
or
sealed
brood.
HopGuard
®
kills
>80%
of
Varroa
in
both
the
large
and
small
honey
bee
colonies
containing
brood,
with
65%
of
the
mites
dying
within
the
first
24
h
post-treatment
[21].
However,
HopGuard
becomes
a
less
effective
mite
treatment
during
the
peak
brood
production
[22].
The
first
objective
of
this
study
was
to
determine,
in
the
laboratory,
the
comparative
toxicities
of
five
acaricidal
products
(Apistan
®
,
Apivar
®
,
Apiguard
®
,
HopGuard
®
,
and
CheckMite
®
)
to
both
the
Varroa
mites
and
host
bees.
The
second
objective
was
to
test
the
acaricidal
efficacies
of
these
same
five
products
as
a
first
hive
treatment,
followed
by
a
second
CheckMite
®
application
to
the
queen-right
colonies
in
the
field.
The
accuracy
of
the
Varroa
populations,
using
the
sugar
shake
method,
was
tested
by
comparing
the
sugar-shake
counts
with
those
of
an
established
protocol,
which
involved
tallying
the
dead
mites
atop
the
hive
bottom
inserts.
2.
Materials
and
Methods
The
laboratory
experiments
tested
the
efficacy
of
the
five
acaricidal
products
(Apistan
®
,
Apivar
®
,
Apiguard
®
,
HopGuard
®
,
and
CheckMite
®
)
in
order
to
kill
the
Varroa
mites
and
to
determine
the
product
toxicities
to
the
worker
honey
bees.
The
products
that
were
widely
used
in
beekeeping
practice
were
also
tested
for
Varroa
suppression
under
field
conditions,
using
fully
developed
queen-right
honey
bee
colonies.
Estimates
of
the
Varroa
infestation
in
the
field
colonies
were
derived
using
two
methods,
namely:
(1)
an
established
protocol
that
involved
counting
the
dead
mites
on
the
hive
bottom
inserts
and
(2)
determining
the
adult
bees'
infestation
using
the
sugar
shake
method.
2.1.
Laboratory
Toxicity
Experiment
In
the
laboratory
studies,
the
bees
were
kept
in
11.4
x
6.3
x
15.2
cm
(W:D:H)
cages,
each
with
a
top,
bottom,
and
sides
made
from
dear
acrylic,
and
a
front,
back,
and
bottom
that
was
constructed
from
a
2.5
mm
wire
screen
mesh.
The
mesh
bottom
insert
was
placed
approximately
2.5
cm
above
the
cage
bottom,
through
which
the
dead
Varroa
mites
could
fall
onto
the
cage
bottom
for
later
collection
and
counting.
The
cages
were
equipped
with
two
60
mL
polypropylene
bottles
(United
States
Plastic
Crop
®
,
Lima,
OH,
USA),
each
with
a
lid
containing
three
small
holes
(Figure
).
One
bottle
was
filled
with
a
(1:1)
sugar
in
water
solution
and
the
other
with
water.
Each
bottle
was
placed
upside
down
in
two
of
the
holes
that
were
drilled
in
the
cage
top,
which
allowed
the
bees
to
feed
from
those
bottles.
The
cage
had
a
hole
drilled
through
each
side,
where
the
rubber
plug
pollen
feeders
(Sigma-Aldrich
®
,
St.
Louis,
MO,
USA)
were
placed.
Lastly,
two
more
small
holes
were
drilled
in
the
acrylic
top
between
the
sugar
and
water
feeder
holes,
and
were
used
to
hang
the
acaricidal
product
with
the
wire
inside
the
cage.
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Laboratory
experiment
Treat
ent
Varroa
count
Air
Water
S
rup
Scree
\
7
CheckMite®
CheckMite®
0.83
g
of
strip
0.42
g
of
strip
(Coumaphos)
(Coumaphos)
Apistan®
I.26
g
of
strip
(Tau-tluvalinate)
Apivar®
2.76
g
of
strip
(Amitraz)
Patty
Apiguard®
0.3
g
of
gel
(Thymol)
/C
-
07
,(ta
HopGuard®
HopGuard
'A
Control
6.65
g
of
strip
3.32
g
of
strip
No
treatment
(Potassium
salt
of
hop
(Potassium
salt
of
hop
beta
Acids)
beta
Acids
X5
Figure
1.
Experimental
design
and
the
application
rates
of
commercial
acaricides
applied
in
each
laboratory
cage.
The
control
treatment
received
no
acaricidal
treatment.
Caged
bees
were
provided
with
sugar
syrup,
water,
and
pollen
patty
Five
replicates
were
carried
out
for
each
treatment,
for
a
total
of
40
cages.
Eight
different
treatment
groups
were
established;
seven
separate
treatment
groups
and
a
non-treated
control
group.
Each
group
consisted
of
five
replicated
cages.
The
caged
worker
bees
of
each
treatment
group
received
a
specific
acaricidal
dose,
and
cages
of
the
control
group
received
no
treatment
(Figure
).
The
eight
groups
(seven
treatment
and
one
control)
consisted
of
the
following:
a
(1)
coumaphos
treatment
group
with
0.83
g
of
a
full
CheckMite
®
strip
(CheckMite
®
1)
(CAS
#
KPOB5KD,
Shawnee
Mission,
KS,
USA);
(2)
coumaphos
treatment
group
with
a
half
dose,
0.42
g
of
a
full
CheckMite
®
strip
(CheckMite
®
1
/
2
);
(3)
Tau-fluvalinate
treatment
group:
1.26
g
(50
x
30
mm)
of
a
full
Apistan
®
strip,
(CAS
#
102851-06-9,
Wellmark
International,
IL,
USA);
(4)
amitraz
treatment
group:
2.76
g
(38
x
40
mm)
of
a
full
Apivar
®
strip
(CAS
#
87243-1,
Veto-pharma,
New
York,
NY,
USA);
(5)
thymol
treatment
group:
0.3
g
of
Apiguard
®
(CAS
#
79671-1,
Vita
Europe
Limited,
Valdosta,
GA,
USA);
(6)
natural
plant
compounds
(hop
acids)
treatment
group:
6.65
g
(70
x
33
mm)
of
full
HopGuard
®
cardboard
strip
(HopGuard
®
1);
(7)
HopGuard
®
treatment
group:
3.32
g
(35
x
33
mm)
of
full
HopGuard
®
cardboard
strip
(HopGuard
®
1
/
2
)
with
the
potassium
salt
of
the
hop
beta
acids
as
the
active
ingredient
(CAS
#
DC301120716Y30816,
Betatec,
Mann
Lake
Ltd.,
Hackensack,
MN,
USA);
and
(8)
a
control
group,
which
received
no
acaricidal
treatment.
The
worker
bees
were
obtained
from
three
Varroa-infested
colonies
from
our
current
stock,
which
was
headed
by
Italian-bred
queens,
Apis
mellifera
ligustica.
The
bees
were
brushed
from
the
brood
frames
into
a
clean
bucket
and
then
randomly
assigned
to
cages
at
a
density
of
—250
bees
per
cage.
The
cages
were
maintained
in
an
unlit
incubator
set
at
a
near
constant
28
°C
and
65%
relative
humidity
values
(RH).
The
caged
bees
were
provided
with
(1:1)
sugar
syrup,
water,
and
pollen
patty,
ad
libitum,
using
the
methods
described
above.
Pieces
of
each
original
acaricidal
product
were
fixed
with
a
wire
and
hung
from
the
inside
of
the
cage
top,
which
ensured
exposure
to
the
bees
from
both
sides,
and
the
amount
of
Apiguard
®
was
placed
in
a
small
plastic
lid
on
the
bottom
mesh
insert,
so
as
to
expose
and
ensure
contact
with
all
of
the
bees
within
the
cage.
The
treatment
dose
of
Apiguard
that
was
applied
to
the
caged
bees
was
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calculated
according
to
the
total
number
of
bees
per
cage
(250),
by
extrapolating
the
data
from
the
treated
colonies
in
a
standard
Langstroth
(LR)
hive
that
received
a
full
dose
for
a
standard
developed
honey
bee
colony,
as
was
advised
by
the
manufacturer.
Similar
to
what
was
done
in
the
full-sized
colonies,
the
acaricidal
products
were
cut
to
a
size
that
ensured
that
the
caged-bees
with
Varroa
contacted
the
exposed
part
of
the
strip
and
received
an
effective
acaricidal
dose.
The
control
group
without
the
acaricidal
treatment
received
the
same
food
as
the
treated
bees
(i.e.,
sugar
solution,
water,
and
pollen
patty).
The
acaricidal
treatments
that
were
administered
to
the
caged-bees
were
meant
to
mimic
those
that
were
applied
to
the
queen-right
honeybee
colonies,
with
the
additional
advantage
of
simultaneously
testing
the
toxicities
on
both
the
mites
and
host
bees.
During
the
experiment,
any
mites
that
were
killed
and
dislodged
by
the
treatment,
fell
through
the
screen
insert
onto
the
bottom
of
the
cage
and
were
then
collected
and
counted
at
6,
24,
and
48
h
intervals.
The
dead
bees
were
then
collected
from
above
the
bottom
screen.
The
counts
of
the
dead
Varroa
and
bees
that
exceeded
the
counts
in
the
control
cages
were
considered
to
have
been
killed
by
the
specific
acaricidal
treatment.
All
of
the
Varroa
that
remained
on
the
adult
bees
throughout
the
test
period
were
considered
to
be
tolerant
of
or
resistant
to
the
treatment.
At
the
end
of
the
experiment
(48
h),
all
of
the
cages
with
the
remaining
bees
were
placed
in
a
freezer
at
—20
°C.
Once
all
of
the
bees
in
a
cage
were
euthanized,
they
were
rinsed
thoroughly
through
a
strainer
and
counted
along
with
their
total
mite
load
(mites
per
cage).
2.2.
Field
Experiment
Using
the
same
acaricidal
products
as
those
from
the
previous
laboratory
experiment,
20
honey
bee
colonies
were
used
to
test
the
Varroa
control
in
the
field.
All
of
the
colonies
that
were
founded
by
Italian
queens,
Apis
mellifera
ligustica,
were
established
in
autumn
2016,
at
an
apiary
that
was
located
at
the
Mississippi
State
University's
Subunit
in
McNeill,
MS
(30°39'46"
N,
89°38'01"
W).
The
experimental
colonies
were
queen-right,
fully
developed,
and
housed
in
10-frame
Langstroth
deep
hive
boxes
(Dadant
&
Sons,
Inc.,
Hamilton,
IL,
USA)
with
plastic
comb
foundations.
Before
the
experiment,
the
colonies
uniformly
occupied
six
to
eight
brood
combs.
The
bees
in
the
experimental
colonies
occupied
all
of
the
spaces
between
the
combs
and
spaces
between
the
hive
wall
and
lateral
comb.
The
Varroa
population
was
estimated
by
counting
the
natural
Varroa
mortality
and
by
performing
the
sugar
shake
diagnostic
method
prior
to
and
after
the
specific
treatments.
The
corrugated
plastic
bottom
boards
(Dadant
&
Sons,
Inc.,
Hamilton,
IL,
USA)
were
inserted
to
capture
dead
Varroa
for
calculating
the
mite
mortality.
Wire
screens
were
installed
above
the
boards
to
prevent
the
bees
from
coming
into
contact
with
the
dead
mites
and
pesticide
contaminated
debris.
On
21
occasions,
from
31
May
to
21
September,
at
3
to
4
day
intervals,
the
dead
mites
were
counted
and
the
bottom
boards
were
cleaned,
Figure
2.
The
levels
of
the
natural
mite
'drop-down'
were
recorded
on
the
first
7
of
these
21
occasions
(sampling
dates),
before
the
treatment
applications.
The
number
of
Varroa
that
were
not
killed
by
the
treatments
was
also
obtained
by
counting
the
Varroa
drop-down
during
the
final
CheckMite
®
treatment
period,
between
18
August
and
21
September,
Figure
.
The
percentage
of
the
Varroa
mites
that
were
killed
(PVK)
by
the
first
treatment
protocol,
using
Apiguard
®
,
Apistan
®
,
Apivar
®
,
and
HopGuard
®
,
was
calculated
using
the
following
equation:
PVK
T1
=
(T1/(T1
+
T2)
x
100)%,
where
(T1)
is
the
number
of
Varroa
killed
by
the
first
treatments
and
(T2)
is
those
killed
by
the
second
CheckMite
®
treatment
[6].
T1
denotes
the
total
number
of
mites
that
dropped
after
the
first
acaricidal
application,
while
T2
denotes
the
number
of
varroa
that
were
collected
after
the
final
CheckMite
®
treatment
on
18
August.
The
treatment
efficacies
were
estimated
by
comparing
the
number
of
Varroa
that
were
killed
before,
and
then
again
after
the
application
of
the
treatment.
The
counts
continued
on
14
sampling
dates
post-application,
from
19
July
to
21
September,
Figure
.
All
of
the
colonies
were
similarly
managed,
until
19
July,
when
they
were
randomly
assigned
to
five
experimental
groups,
each
group
contained
four
colonies
and
each
group
was
assigned
to
one
of
the
five
treatments,
namely:
(1)
Apiguard
®
;
(2)
Apistan
®
;
(3)
Apivar
®
;
(4)
HopGuard
®
;
and
(5)
untreated-control.
All
of
the
treatments
were
applied
to
the
colonies
according
to
their
3
1
May
All
AcaricicIes
lifted
from
colonies
CheckMite®
applied
on
all
colonies
Sugar
Shake
3
Sugar
Shake
2
Sugar
Shake
1
7Augst
18
u
21
September
19
July
A
1
1
11111applied
on
colonies
Insects
2018,
9,
55
5
of
16
respective
manufacturers'
instructions.
Group
1
colonies
each
received
one
tray,
which
contained
50
g
of
Apiguard
®
gel
that
was
placed
on
the
top
bars
of
the
frames.
The
trays
were
replaced
on
31
July
and
were
removed
from
the
colonies
on
18
August.
Group
2
colonies
each
received
two
strips
of
Apistan
®
.
Group
3
colonies
each
received
two
strips
of
Apivar
®
.
Group
4
colonies
each
received
two
strips
of
HopGuard
®
.
Group
5
colonies
were
left
untreated
and
served
as
the
control
group.
Field
experiment
Group:
4
5
.„
-
7
Apiguard®
ApistanC®
Apivar®
1
tray
50
g
2
strips
2
strips
(Thymol)
(Tau-fluvalinate)
(Amitraz)
HopCuard®
2
strips
(Potassium
salt
of
hop
beta
Acids)
Control
No
treatment
No
treatment
Treatments
applied
Final
CheckMite®
treatment
(Natural
varroa
mortality)
(Varroa
killed
by
Acaricides)
(Varroa
left
"Drop-down")
V
Figure
2.
Field
experimental
design
showing
the
acaricidal
products
that
were
tested,
amounts
of
product
used
in
each
honey
bee
colony,
and
the
timeline
detailing
the
products
and
their
application
dates.
All
of
the
Acaricide
strips
and
Apiguard
®
trays
were
removed
from
all
of
the
colonies
on
18
August.
To
evaluate
the
efficiency
of
each
treatment
and
to
quantify
the
Varroa
mite
population
in
each
colony,
a
final
standard
CheckMite
®
treatment
(2
strips/colony)
was
applied
in
each
experimental
colony
on
the
18
August,
Figure
^.
The
Varroa
drops
that
were
as
a
result
of
this
final
treatment,
were
regularly
counted
on
the
hive
bottom
boards,
until
21
September.
To
determine
the
relative
infestation
levels
(number
of
Varroa
mites
per
bee)
in
the
experimental
colonies,
a
sugar
shake
test
[23]
was
conducted
on
the
adult
bees.
For
this
test,
approximately
300-400
worker
honey
bees
were
brushed
into
900
mL
glass
jars
with
a
3.1-mm
mesh
screen
in
their
lids.
Two
tablespoons
of
powdered
sugar
were
added
through
the
screen
and
the
jars
were
rolled,
in
order
to
distribute
the
sugar
evenly
over
the
bees.
After
1
min,
the
jar
and
the
honey
bees
were
vigorously
shaken
over
a
white
paper
plate
for
about
4
min,
and
the
dislodged
Varroa
were
then
counted.
This
method
involved
sampling
the
adult
bees
from
the
middle
brood
frames
at
three
portions
of
the
experiment,
namely:
(1)
at
pre-treatment
on
19
July;
(2)
post-treatment,
right
after
removing
the
four
acaricide
treatments
on
18
August;
and
(3)
the
final
sugar
shake,
which
occurred
after
the
removal
of
the
CheckMite
®
strips
on
21
September,
Figure
.
The
percentage
of
the
Varroa
infestation
was
calculated
based
on
the
number
of
the
Varroa
mites
that
were
found
on
or
removed
from
100
worker
bees.
During
the
sugar
shakes,
the
following
outside
temperatures
(T)
and
relative
humidity
values
(RH)
were
recorded:
19
July:
T
=
32
°C,
RH
=
61%;
18
August:
T =
33
°C,
RH
=
56%;
and
21
September:
T =
30
°C,
RH
=
53%.
X4
Insects
2018,
9,
55
6
of
16
2.3.
Data
and
Statistical
Analyses
The
Varroa
counts
in
the
hive
experiment
were
log
10
-transformed
so
as
to
improve
the
normality
and
to
better
visualize
the
Varroa
population
trends
within
the
honey
bee
colonies.
All
of
the
statistical
analyses
and
figure
generation
were
carried
out
in
the
R
environment
[24].
The
two-way
ANOVA
(analysis
of
variance,
a
=
0.05)
and
Tukey
HSD
test
identified
significant
differences
among
the
treatment
groups
and
dates
within
the
treatment
group.
The
bars
in
the
figures
represent
the
±
standard
error
(±SE).
The
sugar
shake
differences
between
the
Varroa
adult
bee
infestation
before
and
after
the
acaricidal
treatment,
as
well
as
after
the
final
CheckMite
®
treatment,
were
analyzed
and
compared
with
the
relative
cumulative
Varroa
mortalities
after
the
acaricides
treatments,
which
was
followed
by
the
final
CheckMite
®
application.
3.
Results
3.1.
Laboratory
Toxicity
Experiment
The
highest
Varroa
mortality
occurred
for
the
Apivar
®
(91.51%
±
6.45),
HopGuard
®
(91.11%
±
2.63),
and
a
single
Apistan
®
dose
(72.22%
±
4.51),
within
the
first
six
hours
of
exposure.
The
Varroa
mortality
continue
during
24
and
48
h
of
exposure.
The
highest
cumulative
Varroa
mortality
during
the
48
h
of
exposure
was
recorded
in
the
Apivar
®
(98.18%
±
1.8)
and
CheckMite
®
1
(98.03%
±
1.9)
treatment
groups.
The
CheckMite
®
1
also
induced
a
high
bee
mortality
at
the
level
of
59.82%
±
11.86,
in
comparison
with
the
CheckMite
®
1
/
2
dose,
which
induced
a
4.18%
(±1.8)
bee
mortality.
The
Apistan
caused
a
95.72%
(±2.1)
Varroa
moratality.
The
HopGuard
®
1
dose
simultaneously
induced
a
100%
Varroa
mortality
and
a
93.68%
±
4.92
bee
mortality.
On
the
other
hand,
the
HopGuard
®
1
/
2
dose
induced
a
95.79%
(±1.9)
Varroa
mortality
and
a
23.41%
(±6.1)
bee
mortality.
The
Varroa
and
bees
in
cages
that
were
not
exposed
to
an
acaricide
during
the
duration
of
the
experiment
also
died
at
the
levels
of
32.90%
(±4.3)
and
4.12%
(±2.5),
respectively.
Significant
differences
in
the
Varroa
mortalities
were
found
between
the
treatment
groups
(F
=
43.67;
df
=
7;
p
<
0.01;
Figures
3
and
4).
Varroa
Mortality
/
6h
A
x
=
74
1.0
<0
OM
Varroa
Mortality
/
24ti
U
.
o.
'Co
90
e
70
5
8
,;
4
°,
/
•".
v
p
c•Q
IOU
Ci
Treatment
Varroa
Mortality
/
48h
(F.,,,=
13
3,p<0
0011
100
Treatment
Varroa
Remaining
on
Bees
(F,,=
41
n.p<
0
MI)
.;
9
8
'
0
e
iii
7
6
00
50
g
40
0.1
30
til
20
_
ofr
0 /
4,
16
/
iR
Treatment
Treatment
Figure
3.
Varroa
mite
mortality
recorded
in
each
caged-bee
group
at
intervals
of
6,
24,
and
48
h.
Groups
were
exposed
to
a
CheckMite
®
1
dose,
CheckMite
®
V2
dose,
Apistan
®
,
Apivar
®
,
Apiguard
®
,
HopGuard®
1
dose,
HopGuard®
1
/
2
dose,
or
left
untreated
(control
group).
The
term
'remaining'
denotes
the
percentage
of
Varroa
that
survived
after
48
h
of
exposure.
Asterisks
show
ANOVA
significance
levels
for
pairwise
comparisons
between
the
control
and
each
acaricidal
treatment
and
they
are
as
follows:
p
<
0.05
*
and
p
<
0.001
***.
Bars
denote
the
mean
Varroa
mortality
(%)
±
1
standard
error
(SE).
Insects
2018,
9,
55
7
of
16
Overall
Varroa
vs.
Bee
Mortality
%
Varroa,
(F
7.
„=
43.6,
p
<
0.001)
Bee:
(F,
=
38.6.
p
<
0.001)
100
-
a
a
a
a
b
a
,z lli
T
-
r
75
-
a
a
T
Type
50
-
Varroa
a
Bee
25
-
o-
Grz
e6-
Cr
4
1
'
—O
S
‘,&
/
,
§-
6
c.
cz>
,
2
,(9
Treatment
Figure
4.
Total
mortality
for
Varroa
mites
and
their
caged
host
bees
after
48
h
of
exposure
to
1
dose
of
CheckMite
8
,
1
/
2
dose
of
CheckMite
®
,
Apistan
®
,
Apivar
®
,
Apiguard
®
,
1
dose
of
HopGuard
8
,
1
/
2
dose
of
HopGuard®,
and
no
acaricide
(control
group).
Same
letters
indicate
significant
differences
between
a
treatment
and
control
at
cc
=
0.05.
The
only
significant
differences
that
were
detected
occurred
at
p
<
0.001
(***).
Bars
denote
mean
bee
and
mite
mortality
(%)
±
1
standard
error
(SE).
The
mortality
in
the
caged-bees
varied
significantly
among
the
treatments
(F
=
38.63
df
=
7;
and
p
<
0.01).
The
highest
bee
mortality
was
observed
in
the
cages
that
were
treated
with
HopGuard
®
1
dose
and
CheckMite
®
1
dose.
The
lowest
bee
mortalities
occurred
in
the
colonies
that
were
treated
with
the
CheckMite
®
'
dose
4.18%
(±1.8),
Apistan
®
10.61%
(±3.3),
and
Apivar
®
7.13%
(±2.0)
(Figure
).
3.2.
Field
Experiment
Before
the
treatment
on
19
July,
the
20
honey
bee
colonies
had
exhibited
a
relatively
uniform
rate
of
natural
Varroa
mortality,
which
averaged
0.89
±
1.06
mites
per
day
(F
=
0.90,
df
=
4,
p
=
0.48).
During
the
pre-treatment
period,
17.25%
±
4.05
of
the
Varroa
fell
onto
the
bottom
board
inserts.
Three
of
the
four
acaricides
that
were
used
in
the
honey
bee
colonies
(Apiguard
®
,
Apistan
®
,
and
Apivar
®
)
induced
steep
daily
and
monthly
declines
in
the
Varroa
mite
density
within
the
honey
bee
colonies
(p
<
0.05,
Figures
5
and
6).
On
the
other
hand,
the
HopGuard
®
differed
from
the
other
treatments,
in
that
it
had
a
more
uniform
impact
on
the
Varroa
mortality
throughout
the
treatment
period
(p
=
0.9,
Figure
b).
The
Varroa
mortalities
in
the
experimental
groups
were
increased
after
each
specific
acaricide
treatment.
This
was
also
demonstrated
in
the
control
colonies
after
having
received
a
single
CheckMite
treatment,
as
shown
in
Figure
.
The
overall
relative
arroa
mortality
showed
no
significant
differences
among
the
colonies
that
were
exposed
to
the
four
different
acaricides
that
were
used
in
our
study.
However,
the
significant
differences
were
recorded
between
all
of
the
treatment
groups
(F
=
25.8,
df
=
5,
p
<
0.001)
and
both
the
Varroa
mortality
in
the
control,
untreated
colonies,
and
the
natural
Varroa
mortality
(pretreatment),
with
no
differences
being
recorded
between
the
last
two
groups.
Q
tp
Treatment
ApS..
A
20
-
Date:
Apiguard:
12.3,
p=
0.002)
Anis.
10.8,
p
-
0.003)
Control
11.2,
p
-
0.003)
O
5
-
o
-
15
-
Insects
2018,
9,
55
8
of
16
Treatment
Aptanard
IS
00
Con
onISDate
P<0001
P
1\1
1
,11
11N
Treatment
Control
Tre•Intonl
artanntotte
e
e
oat
ale
W!
Natural
Marta
i'1
1
ar
.,
Chtadt$111.
'J
r
1
Nip
Apkrar$Ontat
1
,
0.05
SOON
,
thoslatilta
II NI
11
1\
i
,
II
\:
4
I
I
llooGuardarte:
P
0.9
Checknelee
Treat,.
ill
Treatment
APImr
Figure
5.
Overview
of
the
Varroa
population
dynamics
and
the
Varroa
mortalities
after
specific
acaricide
treatment
within
colonies
of
the
four
treatment
groups
and
control.
Acaricidal
treatments
were
applied
on
19
July
in
all
of
the
groups
except
the
control,
and
a
final
CheckMite
®
treatment
was
applied
in
all
of
the
groups
on
18
August,
so
as
to
kill
the
remaining
mites
in
a
hive.
The
period
between
31
May
and
19
July
is,
for
all
of
the
groups,
a
monitoring
period
to
assess
the
natural
Varroa
mortality,
in
which
no
acaricides
were
applied.
Adult
Bee
Infestation
(%)
Treatmeat:
(F,,,=
33,
p
=
0.016)
Apiguard
Apistan
Apivar
HopGuard
Control
Treatment
Date
July
August
11
September
Figure
6.
Relative
adult
bees
infestations
in
three
time
periods,
determined
using
the
sugar
shake
method.
Adult
bee
infestations
before
the
application
of
four
acaricides,
Apiguard
®
,
Apistan
®
,
Apivar
®
,
and
HopGuard®
(period
before
treatments
from
31
May-19
July,
shown
as
July
in
figure)
and
after
treatments
(19
July-18
August,
shown
as
August
and
September
in
figure,
respectively).
Denote
that
colonies
of
the
control
group
received
the
acaricide
treatment
on
18
August
and
therefore
a
high
Varroa
infestation
was
recorded,
as
August
testing,
just
before
CheckMite®
treatment.
CheckMite®
was
applied
as
a
final
treatment
to
kill
any
remaining
Varroa
mites
in
all
of
the
colonies.
Varroa
infestation
levels
(%
±
SE)
were
calculated
based
on
the
relative
number
of
mites
remaining
on
the
adult
bees
in
each
colony.
The
horizontal
brackets
indicate
the
mean
comparison
between
the
control
and
Apiguard
®
and
Apistan
®
as
a
single
treatment;
*
p
<
0.05;
**
p
<
0.005.
40-
P=0.01
0
30
10-
0-
Insects
2018,
9,
55
9
of
16
The
Varroa
infestation
of
adult
bees
was
estimated
at
6.12%
±
1.93%
in
the
pre-treatment
period,
with
no
significant
differences
in
the
Varroa
among
the
colonies.
Although
the
control
colonies
experienced
a
slight
seasonal
decline
in
Varroa
numbers,
the
sugar
shake
sampling
method
showed
a
much
more
profound
reduction
in
the
mite
infestations
of
the
colonies
that
were
treated
with
Apiguard
®
and
Apistan
®
(p
<
0.05,
Figures
5-7).
The
adult
bees'
infestation,
which
was
detected
with
the
sugar
shake
test
prior
to
the
Apiguard,
Apistan,
Apivar,
and
HopGuard
treatment,
was
4.90%
(±1.2),
5.13%
(±0.9),
7.53%
(±3.0),
and
7.76%
(±2.6),
respectively;
while
the
adult
bee
infestation
after
the
specific
acaricides
treatments
was
0.45%
(±0.09),
2.16%
(±0.62),
0.97%
(±0.5),
and
5.47%
(±1.5),
respectively.
A
significantly
increased
adult
bee
infestation
during
the
treatment
period
was
recorded
in
the
control,
untreated
colonies.
From
the
initial
5.25%
(±1.2),
the
adult
bee
infestation
increased
to
a
15.75%
(±3.4)
infestation.
The
CheckMite
application
induced
reduction
in
the
adult
bee
infestation
was
detected
by
the
sugar
shake
test
in
September,
at
a
level
of
2.34%
(±0.3)
(Figure
4
).
The
sugar
shake
indicated
a
significantly
higher
Varroa
infestation
in
August
(p
<
0.01)
for
the
control
colonies.
The
overall
'sugar
shake
prediction'
estimation
of
the
Varroa
mite
load
that
was
obtained
by
performing
a
sugar
shake
test
in
the honey
bee
colonies,
came
in
full
agreement
with
the
'occurred
Varroa
infestation',
in
terms
of
the
killed
varroa
mites
after
the
CheckMite
®
application,
which
was
obtained
by
counting
the
killed
Varroa
mites
on
the
bottom
boards.
No
significant
differences
in
the
dead
Varroa
were
found
between
the
sugar
shake
prediction
and
the
occurred
Varroa
infestation,
which
were
detected
on
bottom
boards,
except
for
the
control
group.
During
the
CheckMite
®
strips
application
to
the
treatment
groups,
the
relative
efficacies
for
Apiguard
®
,
Apistan
®
,
Apivar
®
,
or
HopGuard
®
were
recorded
at
the
levels
of
86%, 84%,
79%,
and
64%,
respectively.
During
the
same
period
in
the
control,
the
untreated
colonies'
natural
Varroa
mortality
remained
at
the
level
of
11%
(Figure
8).
The
comparative
relative
efficacies
of
the
acaricide
treatments
that
were
conducted
on
the
caged
bees
versus
the
bees
in
the
experimental
colonies
in
the
field
were
found
to
be
the
same
(Figure
).
Overall
Varroa
infestation
%:
Occurred
vs
Predicted
by
Sugar
Shake
Type
Occurred
Infestation
Sugar
Shake
Prediction
Apistan
Apivar
Apiguard
Group
HopGuard
Control
o
Figure
7.
Percentage
of
adult
bees
infested
by
Varroa
mites,
detected
by
sugar
shake
test,
during
the
pre-treatment
period
(July),
after
four
treatment
protocols
(Apiguard
®
,
Apistan
®
,
Apivar
®
,
and
HopGuard®)
(August)
and
final
treatment
of
CheckMite®
(September).
Bars
represent
mean
±
1
SE.
ANOVA
significant
levels
are
as
follows:
p
<
0.05
*.
The
horizontal
brackets
indicate
significant
differences
between
the
actual
and
predicted
levels
of
Varroa
mite
infestation.
T
P=
0.1
P
=
0.06
P
=
0.051
P
=
0.3
P
=
0.004
**
Apis
'
tan
Apivar
Apiguard
HopGuard
Control
Treatment
1
00
-
75
-
(
1
50
-
=
0
25
-
T
Insects
2018,
9,
55
10
of
16
Varroa
Mortality
100
(F
34
=
25.8,
p
<0.001)
T
s
50-
a
40
-
30
-
20
-
10
o-
T
90
80
70
60
G
o
Treatment
Figure
8.
Final
actual
relative
Varroa
mortality
levels
derived
from
the
sugar
shake
method,
compared
with
actual
Varroa
infestation
calculated
from
bottom
board
counts.
The
'actual
varroa
infestation'
was
calculated
based
on
the
total
number
of
Varroa
mites,
counted
from
each
colony
at
the
end
of
the
experiment,
after
the
removal
of
the
CheckMite®
strips.
Denote
that
Varroa
mortality
levels
represent
the
calculated
relative
efficacies
of
acaricides
applied
to
the
honey
bee
colonies
in
the
field.
Overall
Mortality
of
Varroa
in
cage
vs.
in
hive
%
Type
Cage
Hive
Figure
9.
Comparative
relative
efficacy
of
Apiguard
®
,
Apistan
®
,
Apivar
®
,
and
HopGuard
®
applied
to
both
caged
bees
and
honey
bee
colonies,
between
19
July
and
18
August.
All
Varroa
mites
from
the
caged
bees
were
counted
after
48
h
of
exposure
and
after
the
final
CheckMite
®
application
in
colonies.
Bars
indicate
mean
±
1
standard
error
(SE).
ANOVA
significant
level
is
p
<
0.01
**.
Insects
2018,
9,
55
11
of
16
4.
Discussion
The
five
commercial
acaricides
that
were
used
in
our
experiments
demonstrated
the
variable
levels
of
the
Varroa
mite
mortalities,
as
shown
in
the
laboratory
and
field
tests.
However,
the
mite
knockdown
did
vary
among
the
acaricides
during
the
first
48
days
post-treatment.
Apivar
®
,
Apistan
®
,
and
a
full
dose
of
HopGuard
®
killed
most
of
the
Varroa
within
the
first
six
hours
of
exposure,
whereas
the
residual
activity
diminished
rapidly
past
6
h.
In
contrast,
CheckMite
®
and
Apiguard
®
,
for
48
h,
displayed
more
uniform
and
persistent
acaricidal
activity
against
the
mites
on
the
caged
bees.
It
wass
important
to
note
that,
even
after
48
h
of
exposure,
none
of
acaricides
eradicated
all
of
the
Varroa
mites
on
the
caged
honey
bees,
despite
the
naturally
high
mortality
among
the
mites.
In
fact,
after
48
h,
—33%
of
the
Varroa
mites
died
in
the
control
cages.
Coumaphos
in
CheckMite
®
originally
acted
systemically,
whereby
the
bees
consumed
small
quantities
and
spread
the
acaricide
trophalactically.
However,
when
bees'
bodies
had
direct
contact
with
the
coumaphos-impregnated
strips,
more
of
the
acaricide
was
distributed
dermally
among
the
nestmates
[25,26],
thereby
increasing
the
chances
of
the
worker
bees
receiving
an
acute
lethal
dose
(LD
50
=
3
µg
to
6
µg
per
bee).
Within
a
colony,
the
individual
bees
that
were
in
direct
contact
with
the
CheckMite
®
strips
containing
—1300
mg
of
the
active
ingredient,
could
receive
much
higher
coumaphos
doses.
Acaricidal
toxicity
was
more
pronounced
when
the
caged
host
bees
were
kept
in
close
contact
with
the
treatment
strips.
Likewise,
the
bees
in
the
highly
congested
hives
with
less
living
space
might
have
similarly
and
more
frequently
had
with
the
contact
acaricidal
strips.
However,
a
bees'
age,
social
interactions
among
nestmates
and
brood,
food
stores,
and
other
factors
could,
to
some
extent,
limit
the
honey
bees'
exposure
or
sensitivity
to
coumaphos
and
other
neurotoxic
acaricides
[25].
The
additional
bee
exposure
could
have
occurred
as
the
workers
walked
on
and
handled
the
coumaphos-contaminated
wax,
which
could
have
contained
the
coumaphos
residues
from
1.0
µg/kg
to
919
mg/kg
[
].
Other
routes
of
exposure
included
the
bees
feeding
on
honey
or
handling
propolis
[9,28-30].
Clearly,
direct
contact
with
the
acaricidal
strips,
which
contained
a
maximum
dose
of
83
mg
a.i.
coumaphos,
could
have
been
acutely
fatal
to
the
honey
bees,
while
half
of
this
dose
was
largely
benign
to
the
workers.
Lowering
the
coumaphos
dose
by
half
would
have
helped
to
prevent
the
unacceptable
bee
losses,
while
the
active
ingredient
remained
as
effective
as
the
full
dose,
for
killing
the
Varroa.
Thus,
at
appropriate
rates,
the
coumaphos
could
be
regarded
as
safe,
or
at
most,
mildly
toxic
to
honey
bees
[31].
Our
studies
showed
that
some
acute
bee
mortality
could
occur
in
both
the
caged
bees
and
field
colonies
[32],
but
the
losses
could
be
mitigated
with
careful
mite
monitoring
and
strip
dosing.
Apistan
®
was
found
to
be
highly
effective
against
the
Varroa
mites
in
the
caged,
as
well
as
in
the
honey
bee
colonies
experiments.
It
seemed
that
the
newly
established
colonies
that
had
not
been
previously
treated
with
tau-fluvalinate,
carried
Varroa
that
were
sensitive
to
this
active
ingredient.
It
was
therefore
important
to
test
the
acaricide
efficacy
against
the
Varroa
prior
to
the
application
to
the
honey
bee
colonies
in
the
field,
as
resistance
to
Apistan
®
might
have
been
present
in
the
managed
honey
bee
populations
[33]
Similar
to
the
coumaphos
toxicity
to
honey
bees,
mortality
was
observed
in
the
caged
bees
that
were
exposed
to
both
rates
of
HopGuard
®
,
532
mg
per
strip
and
1064
mg
per
strip,
particularly
the
latter.
This
was
expected,
given
that
a
HopGuard
®
rate
of
150
µg/bee
was
deemed
as
safe
for
honey
bees,
and
induced
a
mortality
of
5%
[21].
In
our
experiment,
23
%
of
the
bees
died
after
exposure
to
532
mg
of
beta
acids
on
the
HopGuard
®
(1/2)
strip.
The
bee
mortalities
that
were
observed
after
the
application
of
CheckMite
®
,
full
dose,
or
HopGuard
®
,
full
dose,
to
the
caged
bees,
might
have
varied
in
the
field,
because
of
the
changes
in
the
interior
hive
environment
or
hive
demographics.
Further
research
would
be
needed
in
order
to
assess
the
efficacies
of
the
various
acaricides
throughout
the
reproductive
cycles
of
the
Varroa
mites
and
their
bee
hosts.
Likewise,
we
should
also
identify
any
possible
interactions
that
have
occurred
between
the
acaricides
and
the
climate
(e.g.,
subarctic,
alpine,
temperate,
subtropical,
and
tropical)
in
which
the
colonies
are
managed.
The
bee
mortality
that
was
observed
in
the
laboratory tests
was
far
less
or
absent
in
the
honey
bee
colonies,
except
for
the
HopGuard
®
1
dose
and
HopGuard
®
1
/
2
dose
that
was
applied
in
the
cages
Insects
2018,
9,
55
12
of
16
test,
which
killed
between
23%
and
94%
of
our
bees,
and
was
known
to
be
highly
toxic
to
both
mites
and
honey
bees
[21].
Nevertheless,
the
HopGuard
was
an
efficacious
acaricide
that
killed
>90%
of
the
mites,
and
it
remained
equally
efficacious
after
halving
the
full
test
dose.
This
reduced
rate
of
the
HopGuard
kept
the
bee
mortality
to
an
acceptable
level,
just
slightly
above
the
control
bee
mortality.
Therefore,
we
propose
that
acaricide
treatments,
including
HopGuard,
that
are
applied
at
doses
that
cause
>70%
mite
mortality,
with
less
than
30%
bee
kill,
should
be
considered
as
mite
selective
and
acceptable
for
mite
management
[16].
Apistan
®
,
Apivar
®
strips,
and
Apiguard
®
gel
were
relatively
safe
to
adult
bees,
as
we
recorded
approximately
10%,
7%,
and
5%
bee
mortality,
respectively,
in
our
cages
test.
A
low
bee
mortality
at
the
level
of
4%
was
also
recorded
when
we
used
CheckMite
®
'
dose.
It
was
interesting
that,
by
doubling
the
coumaphos
dose,
the
bee
mortality
was
drastically
increased
by
up
to
60%.
On
the
other
hand,
the
acaricides
Apiguard
®
and
CheckMite
®
1
/
2
dose
were
less
effective
at
killing
the
Varroa,
at
least
when
compared
with
Apistan
®
,
Apivar
®
,
or
HopGuard
®
1
/
2
dose
,in
which
bee
mortality
was
on
tolerable
levels
in
the
cage
experiments.
The
Varroa
mortality
increased
after
Apiguard
®
,
Apistan
®
,
Apivar
®
,
or
HopGuard
®
were
introduced
into
field
colonies
that
contained
brood.
The
relative
efficacy
of
the
Varroa
control
in
the
honey
bee
colonies,
using
the
previously
mentioned
acaricides,
Apiguard
®
,
Apistan
®
,
Apivar
®
,
or
HopGuard
®
,
which
were
performed
between
19
July
and
21
September,
as
were
determined
by
using
the
sugar
shake
method
and
calculating
the
Varroa
mortalities
that
were
detected
on
the
hives'
bottom
boards
in
our
experiment
was
86%,
84%,
79%,
and
64%,
respectively.
The
natural
Varroa
mortality
during
the
pre-treatment
period
of
49
days
was
less
than
one
mite
per
day,
and
during
the
pretreatment
period.
The
natural
mite
mortality
in
the
control
(untreated)
colonies
rose
to
an
average
of
3.7
mites/day,
as
detected
on
the
bottom
boards.
The
final
CheckMite
®
treatment
that
was
completed
on
the
previous
control
colonies,
ensured
that
these
colonies
survived,
particularly
given
that
the
unchecked
Varroa
populations
continued
to
rise
before
the
CheckMite
®
strips
were
installed.
It
was
therefore
evident
that
the
sugar
shake
test
revealed
that
approximately
16%
of
the
adult
bees
in
the
untreated
control
colonies
were
infested
prior
to
the
CheckMite
®
application.
After
treatment,
the
adult
bee
infestation
was
reduced
to
2%.
Amitraz,
as
the
active
substance
(formamidine)
in
the
Apivar
®
product,
was
an
effective
acaricide,
based
on
our
laboratory
tests
with
the
caged
bees
that
were
infested
with
Varroa.
It
was
one
of
the
first
chemicals
that
was
tested
in
1979
for
the
control
of
the
Varroa
mite
in
the
managed
honey
bee
colonies
[34].
In
previous
years,
the
reports
of
the
variable
efficacies
of
Amitraz,
in
order
to
control
the
Varroa,
indicated
that
the
mite
populations
were
becoming
resistant
[35-37].
Mite
resistance
to
Amitraz
around
the
globe
varied
considerably
from
0%
to
96%,
depending
on
the
acaricide
management
that
was
adopted
[38,39].
Our
populations
of
Varroa
mites
appeared
highly
susceptible
to
Amitraz
(Apivar
®
)
and
this
product
was
highly
effective
for
controlling
the
Varroa
mites
in
both
the
cage
and
field
experiments,
with
mean
mortalities
of
—98%
and
—79%,
respectively.
A
lower
efficacy
was
established
in
the
colonies
after
using
the
coumaphos
treatment
and
counting
the
dead
Varroa
that
were
on
the
bottom
boards,
in
comparison
to
the
estimated
efficacy
using
the
sugar
shake,
and
the
survived
Varroa
were counted
on
the
adult
bees.
Our
efficacy
values
for
the
Apivar
that
was
obtained
in
the
cages
test
concurred
with
those
that
were
derived
in
different
studies,
namely:
78.8-87.3%
[38],
98.4-99.5%,
[40],
and
<60.1%
[41].
Different
degrees
of
efficacy
had
been
established
for
Apiguard
®
.
In
one
experiment,
the
mortality
rate
of
the
Varroa
mite
after
the
Apiguard
®
treatments
was
86%,
whereas,
in
others,
the
natural
mortality
rate
in
the
control
colonies
was
approximately
23%
[19].
It
was
also
interesting
that
the
46%
efficacy
of
the
Apiguard
®
in
the
colonies
with
brood
in
the
continental
climatic
conditions
['n]
was
lower
than
our
current
findings.
Different
climatic
and
geographic
conditions,
as
well
as
hive
management
systems
may
affect
Apiguard
®
efficacy
against
varroa
[2,42].
Perhaps,
Apiguard
®
efficacy,
increased
when
they
were
applied
to
the
colonies
that
were
managed
in
a
subtropical
climate.
It
was
noteworthy
that
the
Apiguard
®
treatments
that
were
conducted
in
autumn
had
little
to
no
negative
effect
on
the
early
spring
bee
populations
[
Insects
2018,
9,
55
13
of
16
The
results
of
this
study
gave
an
indication
of
the
limited
effect
of
HopGuard
®
as
a
long-term
or
persistent
organic
treatment
in
honey
bee
colonies,
in
comparison
to
the
other
acaricides
that
were
tested.
The
effectiveness
of
HopGuard
®
in
colonies
with
brood
lasted
about
seven
days.
Therefore,
to
extend
the
residual
activity
of
HopGuard
®
in
the
colonies
with
brood,
three
consecutive
HopGuard
®
treatments
should
have
been
applied
[
].
This
organic
treatment
thus
effectively
contributed
to
the
Varroa
reduction
during
the
season
and,
coupled
with
other
control
treatment
applications
during
late
summer
and
autumn,
could
reduce
an
infestation
to
an
almost
negligible
level,
which
was
confirmed
by
the
sugar
shake
test
that
was
completed
in
September,
which
showed
the
low
infestation
levels
in
the
colonies.
The
higher
efficacy
in
killing
the
Varroa
mites
occurred
after
late
summer.
For
example,
the
September
treatments
induced
a
higher
mite
mortality
when
compared
with
the
earlier
treatments,
because
of
a
larger
proportion
of
more
susceptible
phoretic
Varroa
on
adult
bees
[
].
We
also
confirmed
the
high
efficacy
of
HopGuard
®
against
the
Varroa
mites
on
adult
caged
bees.
Therefore,
this
product
could
be
applied
to
colonies
with
or
without
brood
[
].
The
efficacy
of
the
treatments
could
have
been
underestimated
because
of
the
reproduction
of
the
surviving
Varroa
and
the
mite
re-invasions
of
the
experimental
colonies
[
].
Alternatively,
the
Varroa
mortalities
might
have
have
been
overestimated
when
the
CheckMite
®
was
applied
as
a
final
treatment
in
the
colonies,
which
was
less
than
100%
effective
in
late
September.
Throughout
the
season,
all
of
the
treated
and
control
colonies
developed
normally
and
remained
strong
without
any
mass
deaths
occurring
among
the
worker
bees
or
queens
[46,47].
It
was
noteworthy
that
the
efficacy
of
controlling
the
Varroa
in
cages
corresponded
to
the
efficacy
of
the
Varroa
control
in
the
colonies.
The
rapid
growth
of
the
mite
populations
in
the
untreated
control
colonies
showed
that
the
low
mite
populations
in
the
spring
and
early
summer
did
not
always
remain
low,
even
when
the
honey
bee
populations
began
to
decline
in
the
fall
and
winter.
Therefore,
sampling
bees
throughout
the
season,
to
count
mites,
or
performing
the
sugar
shake
test,
to
remove
the
mites
for
counting,
might
be
required
in
order
to
ensure
the
seasonal
control
of
the
Varroa
with
>70%
efficacy
[23,48].
The
sugar
shake
test,
which
was as
effective
at
counting
the
Varroa
on
the
hive
bottoms,
was
a
more
convenient
test
for
monitoring
the
Varroa
populations
and
for
assessing
the
acaricide
effectiveness.
An
accurate
diagnosis
ensured
a
good
projection
of
a
colony's
real
infestation
level,
and
assisted
in
determining
the
potential
mite
population
growth
in
the
colonies.
A
powdered
sugar
shake,
followed
by
an
acaricide
treatment,
organic
or
conventional,
could
be
used
to
diagnose
and
treat
Varroa
promptly.
5.
Conclusions
Our
results
show
that
various
acaricides
are
variably
effective
for
Varroa
suppression
when
the
mite
populations
are
rising
and
brood
is
present.
Hence,
colonies
that
are
treated
with
HopGuard
®
in
summer
will
require
another
treatment
in
autumn,
so
as
to
prevent
a
Varroa
resurgence.
When
there
are
smaller
host
broods
within
the
honey
bee
colonies,
both
the
conventional
and
organic
acaricides
are
effective
Varroa
controls.
However,
the
control
must
be
preceded
by
an
accurate
Varroa
diagnoses,
in
order
to
maintain
the
mite
levels
below
an
economic
threshold
that
is
needed
for
the
colony's
survival.
Author
Contributions:
A.G.,
and
M.A.
designed
the
experiment.
A.G.,
P.R.K.
and
J.A.
conducted
the
experiments.
M.A.
and
A.G.
carried
out
the
statistical
analyses.
A.G.
and
M.A.
wrote
the
manuscript;
J.A.
and
B.S.
revised
it.
All
authors
approve
the
content
and
results
of
this
study.
Acknowledgments:
We
are
grateful
for
the
assistance
provided
by
Chris
Werle
and
Janie
Ross
throughout
the
experiments
and
with
data
collection,
and
to
Janie
Ross
for
the
English
editing.
The
research
was
partly
financially
supported
by
the
Slovenian
Research
Agency,
Research
Program
P4-133.
Conflicts
of
Interest
The
authors
declare
no
conflicts
of
interest
Insects
2018,
9,
55
14
of
16
References
1.
Anderson,
D.L.;
Trueman,
J.W.
Varroa
jacobsoni
(acari:
Varroidae)
is
more
than
one
species.
Exp.
Appl.
Acarol.
2000,
24,
165-189.
[CrossRef]
[PubMed]
2.
Rosenkranz,
P.;
Aumeier,
P.;
Ziegelmann,
B.
Biology
and
control
of
Varroa
destructor.
J.
Invertebr.
Pathol.
2010,
103
(Suppl.
1),
S96—S119.
[CrossRef]
[PubMed]
3.
Miozes-Koch,
R.;
Slabezki,
Y.;
Efrat,
H.;
Kalev,
H.;
Kamer,
Y.;
Yakobson;
Dag,
A.
First
detection
in
israel
of
fluvalinate
resistance
in
the
varroa
mite
using
bioassay
and
biochemical
methods.
Exp.
Appl.
Acarol.
2000,
24,35-43.
[CrossRef]
[PubMed]
4.
Floris,
I.;
Satta,
A.;
Cabras,
P.;
Garau,
V.L.;
Angioni,
A.
Comparison
between
two
thymol
formulations
in
the
control
of
Varroa
destructor:
Effectiveness,
persistence,
and
residues.
J.
Econ.
Entomol.
2004,
97,187-191.
[CrossRef]
[PubMed]
5.
Spreafico,
M.;
Eordegh,
F.R.;
Bernardinelli,
I.;
Colombo,
M.
First
detection
of
strains
of
Varroa
destructor
resistant
to
coumaphos.
Results
of
laboratory
tests
and
field
trials.
Apidologie
2001,
32,49-55.
[CrossRef]
6.
Gregorc,
A.;
Poldukar,
J.
Rotenone
and
oxalic
acid
as
alternative
acariddal
treatments
for
Varroa
destructor
in
honeybee
colonies.
Vet.
Parasitol.
2003,
111,
351-360.
[CrossRef]
7.
Melathopoulos,
A.P.;
Gates,
J.
Comparison
of
two
thymol-based
acaricides,
api
life
var
(R)
and
apiguard
(TM),
for
the
control
of
varroa
mites.
Am.
Bee
J.
2003,
143,
489-493.
8.
Bogdanov,
S.;
Kilchenmann,
V.;
Imdorf,
A.;
Fluri,
P.
Residues
in
honey
after
application
of
thymol
against
varroa
using
the
frakno
thymol
frame.
Am.
Bee
J.
1998,
138,
610-611.
9.
Wanner,
K.
Varroacides
and
their
residues
in
bee
products.
Apidologie
1999,
30,
235-248.
[CrossRef]
10.
Baxter,
J.;
Ellis,
M.;
Wilson,
W.
Field
evaluation
of
apistan
and
five
candidate
compounds
for
parasitic
mite
control
in
honey
bees.
Am.
Bee
J.
2000,
11,
898-900.
11.
Elzen,
P.J.;
Baxter,
J.R.;
Spivak,
M.;
Wilson,
W.T.
Amitraz
resistance
in
varroa:
New
discovery
in
North
America.
Am.
Bee
J.
1999,
139,
362-362.
12.
Elzen,
P.J.;
Westervelt,
D.
Detection
of
coumaphos
resistance
in
Varroa
destructor
in
florida.
Am.
Bee
J.
2002,
142,291-292.
13.
Gracia-Salinas,
M.J.;
Ferrer-Dufol,
M.;
Latorre-Castro,
E.;
Monero-Manera,
C.;
Castillo-Hernandez,
J.A.;
Lucientes-Curd,
J.;
Peribanez-Lopez,
M.A.
Detection
of
fluvalinate
resistance
in
Varroa
destructor
in
spanish
apiaries.
J.
Apic.
Res.
2006,
45,101-105.
[CrossRef]
14.
Bak,
B.;
Wilde,
J.;
Siuda,
M.
Characteristics
of
north-eastern
population
of
Varroa
destructor
resistant
to
synthetic
pyrethroids.
Med.
Weter.
2012,
68,
603-606.
15.
Thompson,
H.M.;
Brown,
M.A.;
Ball,
R.F.;
Bew,
M.H.
First
report
of
Varroa
destructor
resistance
to
pyrethroids
in
the
UK.
Apidologie
2002,
33,357-366.
[CrossRef]
16.
Lindberg,
C.M.;
Melathopoulos,
A.P.;
Winston,
M.L.
Laboratory
evaluation
of
miticides
to
control
Varroa
jacobsoni
(acari:
Varroidae),
a
honey
bee
(hymenoptera:
Apidae)
parasite.
J.
Econ.
Entomol.
2000,
93,
189-198.
[CrossRef]
[PubMed]
17.
Gregorc,
A.;
Jelenc,
J.
Control
of
Varroa
jacobsoni
oud.
In
honeybee
colonies
using
apilife-VAR.
Zb.
Vet.
Fak.
Univ.
Ljublj.
1996,
33,
231-235.
18.
Fassbinder,
C.;
Grodnitzky,
J.;
Coats,
J.
Monoterpenoids
as
possible
control
agents
for
Varroa
destructor.
J.
Apic.
Res.
2002,
41,83-88.
[CrossRef]
19.
Mattila,
H.R.;
Otis,
G.W.
The
efficacy
of
apiguard
against
varroa
and
tracheal
mites,
and
its
effect
on
honey
production:
1999
trial.
Am.
Bee
J.
2000,
140,
969-973.
20.
Gregorc,
A.;
Planinc,
I.
The
control
of
Varroa
destructor
in
honey
bee
colonies
using
the
thymol-based
acaricide-apiguard.
Am.
Bee
J.
2005,
145,
672-675.
21.
Rademacher,
E.;
Marika,
H.;
Saskia,
S.
The
development
of
hopguard®
as
a
winter
treatment
against
Varroa
destructor
in
colonies
of
Apis
mellifera.
Apidologie
2015,
6,748-759.
[CrossRef]
22.
DeGrandi-Hoffman,
G.;
Ahumada,
F.;
Curry,
R.;
Probasco,
G.;
Schantz,
L.
Population
growth
of
Varroa
destructor
(acari:
Varroidae)
in
commercial
honey
bee
colonies
treated
with
beta
plant
acids.
Exp.
Appl.
Acarol.
2014,
64,171-186.
[CrossRef]
[PubMed]
23.
Gregorc,
A.;
Knight,
P.R.;
Adamczyk,
J.
Powdered
sugar
shake
to
monitor
and
oxalic
acid
treatments
to
control
varroa
mites
(Varroa
destructor
anderson
and
trueman)
in
honey
bee
(Apis
mellifera)
colonies.
J.
Apic.
Res.
2017,
56,
71-75.
[CrossRef]
Insects
2018,
9,
55
15
of
16
24.
R
Core
Team.
R:
A
Language
and
Environment
for
Statistical
Computing;
R
Foundation
for
Statistical
Computing:
Vienna,
Austria,
2011.
25.
Van
Buren,
N.W.M.;
Marien,
A.G.H.;
Velthuis,
H.H.W.
The
role
of
trophallaxis
in
the
distribution
of
perizin
in
a
honeybee
colony
with
regard
to
the
control
of
the
varroa
mite.
Entomol.
Exp.
Appl.
1992,
65,157-164.
[CrossRef]
26.
Van
Buren,
N.W.M.;
Marien,
A.G.H.;
Velthuis,
H.H.W.
The
effectiveness
of
systemic
agents
used
to
control
the
mite,
Varroa
jacobsoni,
in
colonies
of
the
honey
bee,
Apis
mellifera
depends
on
food
distribution
patterns.
Apidologie
1993,
24,33-43.
[CrossRef]
27.
Mullin,
C.A.;
Frazier,
M.;
Frazier,
J.L.;
Ashcraft,
S.;
Simonds,
R.;
Vanengelsdorp,
D.;
Pettis,
J.S.
High
levels
of
miticides
and
agrochemicals
in
North
American
apiaries:
Implications
for
honey
bee
health.
PLoS
ONE
2010,
5,
e9754.
[CrossRef]
[PubMed]
28.
Tremolada,
P.;
Bernardinelli,
I.;
Colombo,
M.;
Spreacfico,
M.;
Vighi,
M.
Coumaphos
distribution
in
the
hive
ecosystem:
Case
study
for
modelling
applications.
Ecotoxicology
2004,13,589-601.
[CrossRef]
[PubMed]
29.
Martell,
A.-C.;
Zeggane,
S.;
Aurieres,
C.;
Drahnudel,
P.;
Faucon,
J.-P.;
Aubert,
M.
Acaricide
residues
in
honey
and
wax
after
treatment
of
honey
bee
colonies
with
apivar
®
or
asuntol
®
50.
Apidologie
2007,
6,
534-544.
[CrossRef]
30.
Premrov
Bajuk,
B.;
Babnik,
K.;
Snoj,
T.;
Mainski,
L.;
Pislak
Ocepek,
M.;
gkof,
M.;
Jen6e',
V.;
Filazi,
A.;
gtajnbaher,
D.;
Kobal,
S.
Coumaphos
residues
in
honey,
bee
brood,
and
beeswax
after
Varroa
treatment.
Apidologie
2017,
48,
588-598.
[CrossRef]
31.
Extension
Toxicology
Network.
Pesticide
Information
Prol3le:
Coumaphos.
Extoxnet.
2001.
Available
online:
http://extoxnet.orst.edu/pips/coumapho.htm
(Accessed
on
10
December
2015).
32.
Gregorc,
A.
A
clinical
case
of
honey
bee
intoxication
after
using
coumaphos
strips
against
Varroa
destructor.
J.
Apic.
Res.
2012,
51,
142-143.
[CrossRef]
33.
Koumad,
S.;
Haddad,
N.
Resistance
of
Varroa
destructor
to
apistan©
and
bayvarol.
J.
Zool.
Res.
2015,
1,35-42.
34.
Merrington,
0.
Bibliography
on
the
Use
of
Amitraz
for
Varroa
Control
in
bees
(Apis
Spp.)
(1979-1989);
Cambridge
Animal
and
Public
Health
Ltd.:
Cambridge,
UK,
1990;
p.
36.
35.
Maggi,
M.D.;
Ruffinengo,
S.R.;
Negri,
P.;
Eguaras,
M.J.
Resistance
phenomena
to
amitraz
from
populations
of
the
ectoparasitic
mite
Varroa
destructor
of
Argentina.
Parasitol.
Res.
2010,
107,
1189-1192.
[CrossRef]
[PubMed]
36.
Milani,
N.
The
resistance
of
Varroa
jacobsoni
oud.
To
acaricides.
Apidologie
1999,
30,
229-234.
[CrossRef]
37.
Al
Toufailia,
H.M.;
Ratnieks,
F.L.W.
How
effective
is
apistan
at
killing
Varroa?
Bee
Craft
2016,
98,
7-11.
38.
Floris,
I.;
Cabras,
P.;
Garau,
V.L.;
Minelli,
E.V.;
Satta,
A.;
Troullier,
J.
Persistence
and
effectiveness
of
pyrethroids
in
plastic
strips
against
Varroa
jacobsoni
(acari:
Varroidae)
and
mite
resistance
in
a
mediterranean
area.
J.
Econ.
Entomol.
2001,
94,
806-810.
[CrossRef]
[PubMed]
39.
Al
Naggar,
Y.;
Tan,
Y.;
Rutherford,
C.;
Connor,
W;
Griebel,
P.;
Giesy,
J.P.;
Robertson,
A.J.
Effects
of
treatments
with
apivar((r))
and
thymovar((r))
on
v-destructor
populations,
virus
infections
and
indoor
winter
survival
of
canadian
honey
bee
(Apis
mellifera
L.)
colonies.
J.
Apic.
Res.
2015,
54,
548-554.
[CrossRef]
40.
Vallon,
J.;
Salvary,
F.;
Jourdan,
P.
Suivi
de
l'efficacite
des
traitements
contre
Varroa
destructor
beneficiant
d'une
amm
au
cours
de
l'automne
et
l'hiver
2006/2007.
Bull.
Tech.
Apic.
2007,
2,49-54.
41.
Pires,
S.;
Murilhas,
A.;
Pereira,
O.;
Maia,
M.
Current
Effectiveness
of
Amitraz
against
Varroa
in
Portugal.
In
Proceedings
of
the
39th
Apimondia
International
Apicultural
Congress,
Dublin,
Ireland,
21
November
2005.
42.
Lodesani,
M.;
Costa,
C.
Maximizing
the
efficacy
of
a
thymol
based
product
against
the
mite
Varroa
destructor
by
increasing
the
air
space
in
the
hive.
J.
Apic.
Res.
2008,
47,113-117.
[CrossRef]
43.
Degrandi-Hoffman,
G.;
Ahumada,
F.;
Probasco,
G.;
Schantz,
L.
The
effects
of
beta
acids
from
hops
(Humulus
lupulus)
on
mortality
of
Varroa
destructor
(acari:
Varroidae).
Exp.
Appl.
Acarol.
2012,
58,
407-421.
[CrossRef]
[PubMed]
44.
Wilkinson,
D.;
Smith,
G.C.
A
model
of
the
mite
parasite,
Varroa
destructor,
on
honeybees
(Apis
mellifera)
to
investigate
parameters
important
to
mite
population
growth.
Ecol.
Model.
2002,
148,
263-275.
[CrossRef]
45.
Frey,
E.;
Schnell,
H.;
Rosenkranz,
P.
Invasion
of
Varroa
destructor
mites
into
mite-free
honey
bee
colonies
under
the
controlled
conditions
of
a
military
training
area.
J.
Apic.
Res.
2011,
50,
138-144.
[CrossRef]
46.
Imdorf,
A.;
Charriere,
J.D.;
Bachofen,
B.
Efficiency
checking
of
the
Varroa
jacobsoni
control
methods
by
means
of
oxalic
acid.
Apiacta
1997,
3,
89-91.
Insects
2018,
9,
55
16
of
16
47.
Higes,
M.;
Meana,
A.;
Suarez,
M.; Llorente,
J.
Negative long-term
effects
on
bee
colonies
treated
with
oxalic
acid
against
Varroa
jacobsoni
oud.
Apidologie
1999,
30,
289-292.
[CrossRef]
48.
Fakhimzadeh,
K.;
Ellis,
J.D.;
Hayes,
J.W.
Physical
control
of
varroa
mites
(Varroa
destructor):
The
effects
of
various
dust
materials
on
varroa
mite
fall
from
adult
honey
bees
(Apis
mellifera)
in
vitro.
J.
Apic.
Res.
2011,
50,
203-211.
[CrossRef]
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