Effect of Bacillus sphaericus strain SSII-1 on honey bees, Apis mellifera


Davidson, E.W.; Morton, H.L.; Moffett, J.O.; Singer, S.

Journal of Invertebrate Pathology 29(3): 344-346

1977


When mosquito-larvicidal B. sphaericus strain SSII-1 cultures were fed to newly emerged adult honey bees and to bee colonies, no effect was found on the longevity of newly emerged bees nor on the brood production of colonies.

JOURNAL
OF
INVERTEBRATE
PATHOLOGY
29,
344-346
(1977)
Effect
of
Bacillus
sphaericus
Strain
SSII-1
on
Honey
Bees,
Apis
melliferal
ELIZABETH
WEST
DAVIDSON,
*
HOWARD
L.
MORTON,t
JOSEPH
0.
MOFFETT,t
AND
SAMUEL
SINGERt
*
Department
of
Zoology,
Arizona
State
University,
Tempe,
Arizona
85281;
t
Agricultural
Research
Service,
U.S.
Department
of
Agriculture,
2000
East
Allen
Road,
Tucson,
Arizona
85719;
and
t
Department
of
Biological
Sciences,
Western
Illinois
University,
Macomb,
Illinois
61455
Received
May
12,
1976
When
mosquito
-larvicidal
B.
sphaericus
strain
SSII-1
cultures
were
fed
to
newly
emerged
adult
honey
bees
and
to
bee
colonies,
no
effect
was
found
on
the
longevity
of
newly
emerged
bees
nor
on
the
brood
production
of
colonies.
Bacillus
sphaericus
strain
SSII-1
is
in-
secticidal
for
mosquito
larvae
and
will
be
fi
eld-tested
as
a
microbial
control
agent.
We
report
results
of
research
to
determine
the
effects
of
this
bacterium
on
the
longevity
and
reproduction
of
an
important
nontarget
in-
sect,
the
honey
bee,
Apis
mellifera.
MATERIALS
AND
METHODS
Bacterial
cultures.
Bacillus
sphaericus
strain
SSII-1
and
noninsecticidal
strain
ATCC
7054,
both
var.
fustformis,
were
cul-
tured
in
synthetic
liquid
medium
to
produce
synchronously
growing
fi
nal
whole
cultures
(FWC)
at
18-24
hr
after
inoculation
(Singer
et
al.,
1966;
Singer,
1974).
Final
whole
cul-
tures
were
diluted
with
20%
sucrose
for
feeding
to
bees.
Insecticidal
activity
and
bacterial
viability
were
preserved
when
FWC
were
diluted
with
20%
sucrose
solu-
tion,
but
not
when
diluted
with
60%
sucrose.
Longevity
studies.
Five
2
x
6
x
6
-in.
cages,
each
containing
10
g
of
newly
emerged
bees,
were
used
for
each
bacterial
culture
at
each
concentration
[1
part
FWC/
100
parts
20%
sucrose
(10
-2
)
and
1
part
FWC/10,000
parts
20%
sucrose
(10')]
and
for
20
and
60%
sucrose
control
solutions
1
Mention
of
a
proprietary
product
or
company
name
does
not
constitute
an
endorsement
of
this
product
by
the
U.S.
Department
of
Agriculture.
containing
no
bacterial
culture.
Five
grams
of
maintenance
diet,
consisting
of
11
parts
pollen,
10
parts
sucrose,
9
parts
Drivert
(92%
sucrose,
8%
invert
sugar;
California
and
Hawaii
Sugar
Co.),
was
added
at
the
beginning
of
the
test.
Distilled
water
and
diluted
bacterial
culture
or
sucrose
solution
were
made
available
to
bees
in
5
-dram
plas-
tic
vials
and
were
replenished
daily.
Dead
bees
were
removed
daily,
counted,
and
frozen.
These
methods
have
been
used
previously
in
herbicide
studies
(Morton
et
al.,
1972)
and
in
evaluating
the
effects
of
alfalfa
looper
nuclear
polyhedrosis
virus
on
honey
bees
(Morton
et
al.,
1975).
Longevity
studies
took
place
in
Tucson,
Arizona,
be-
tween
March
4
and
May
4,
1975.
Colonies
in
reproduction
studies
were
held
in
fl
ight
cages
in
Tucson
from
March
28
to
May
2,
1975,
when
they
were
moved
to
a
desert
apiary
where
they
were
observed
until
July
10,
1975.
Reproduction
studies.
Honey
bee
colo-
nies
were
initiated
from
3
lb
of
bees
and
a
laying
queen.
Following
a
free
-flight
period
of
4
days,
each
colony
was
moved
into
a
12
x
12
x
10
-ft
Saran
-mesh
fl
ight
cage.
Each
bacterial
culture
was
fed
at
10
-2
con-
centration
to
three
caged
colonies,
as
was
the
20%
sucrose
control.
Solutions
were
fed
from
1
-pint
jars
placed
directly
above
the
frame
containing
the
brood
and
were
re
-
344
Copyright
1977
by
Academic
Press,
Inc.
All
rights
of
reproduction
in
any
form
reserved.
ISSN
0022-2011
BACILLUS
SPHAERICUS
AND
HONEYBEES
345
plenished
daily.
After
28
days
of
confine-
ment
within
fl
ight
cages,
colonies
were
moved
to
an
apiary
where
they
were
allowed
to
free
-fly
and
collect
pollen
and
nectar,
though
bacterial
culture
was
still
replenished
weekly
for
4
weeks.
At
weekly
intervals,
brood
development
was
observed,
and
samples
of
larvae
and
pupae
were
removed
and
frozen.
The
area
of
the
capped
brood
was
measured
21,
28,
and
56
days
after
bacterial
cultures
were
fi
rst
introduced.
These
methods
are
similar
to
those
de-
scribed
by
Morton
and
Moffett
(1972).
Reisolation
of
B.
sphaericus.
Dead
frozen
adult
bees
from
longevity
tests
and
larvae
and
pupae
from
reproduction
tests
were
sur-
face
-sterilized
by
being
dipped
into
70%
ethanol
for
30
sec.
They
were
then
rinsed
in
sterile
water
and
crushed
in
25
ml
of
syn-
thetic
liquid
bacterial
medium
in
125
-ml
Erlenmeyer
fl
asks.
The
fl
asks
were
incu-
bated
at
28°C
for
24
hr
in
an
orbital
water
-
bath
shaker
at
100
rpm.
Resulting
cultures
were
diluted
and
plated
on
brain
-heart
in-
fusion
agar
plates
(Difco,
Detroit,
Michigan)
and
were
incubated
for
24
hr
at
32°C.
Bac-
terial
colonies
were
then
selected
on
the
basis
of
similarity
of
colony
appearance
to
stock
cultures
of
B.
sphaericus.
Colonies
were
confirmed
as
B.
sphaericus
by
the
presence
of
round
terminal
or
subterminal
spores
swelling
the
sporangium
(Gordon
et
al.,
1973)
in
smears
stained
with
malachite
green
and
safranin
(Conn
et
al.,
1957).
After
restreaking
to
establish
purity,
B.
sphaeri-
cus
colonies
were
inoculated
into
liquid
synthetic
medium,
and
cultures
were
brought
to
synchrony.
FWC,
18
to
24
hr
old,
were
bioassayed
against
second-instar
larvae
of
the
mosquito
Culex
pipiens
quinquefasciatus
.
Values
for
LD„,
based
on
dilution
of
FWC
(Singer,
1974),
were
compared
with
stock
laboratory
cultures
of
SSII-1
and
ATCC
7054.
RESULTS
AND
DISCUSSION
Bacillus
sphaericus
was
isolated
from
adult
bees
fed
SSII-1
and
ATCC
7054
in
longevity
tests.
When
bioassayed,
B.
sphaericus
cultures
isolated
from
bees
fed
SSII-1
gave
LD„
values
of
approximately
1
part
culture/100,000
parts
water
(10
-
s),
similar
to
values
found
with
stock
cultures
of
SSII-1.
Bacillus
sphaericus
cultures
iso-
lated
from
bees
fed
ATCC
7054
were
not
pathogenic
to
mosquito
larvae.
No
B.
sphaericus
was
isolated
from
bees
fed
60%
sucrose;
however,
mosquito
-larvicidal
B.
sphaericus,
presumed
to
be
strain
SSII-1,
was
isolated
from
controls
fed
20%
sucrose,
indicating
contamination
of
the
20%
sucrose
control.
No
B.
sphaericus
was
isolated
from
larvae
or
pupae
from
bee
colonies
in
the
reproduction
tests.
In
longevity
tests,
the
half-life
was
defined
as
the
number
of
days
required
for
one-half
of
the
bees
in
a
cage
to
die.
Half-lives
of
treated
groups
did
not
differ
significantly
from
the
60%
control
except
for
those
fed
ATCC
7054
at
10
-2
dilution
at
the
5%
level
of
probability,
as
calculated
by
Dun
-
can's
multiple
-range
test
(Table
1).
In
reproduction
tests,
eggs
were
observed
in
all
but
one
of
the
colonies
when
they
were
moved
inside
the
fl
ight
cages
and
fi
rst
fed
the
bacterial
cultures
or
sucrose
solution.
TABLE
1
HALF-LIFE
OF
NEWLY
EMERGED
ADULT
HONEY
BEES
FED
BACTERIAL
CULTURES
IN
20%
SUCROSE
AND
20
AND
60%
SUCROSE
CONTROLS'
Bacterial
culture
Concentra-
tion
in
20%
Half
-
sucrose
lifeb
B.
sphaericusISSII-1
B.
sphaericusISSII-1
B.
sphaericus/ATCC
7054
B.
sphaericuslATCC
7054
20%
Sucrose
control'
60%
Sucrose
control
10
-4
10
-2
10
-'
10
-2
0
0
53
ab
48
ab
53
ab
46
b
52
ab
57
a
a
Half-life
is
number
of
days
required
for
one-half
of
the
bees
in
a
cage
to
die.
6
Means
followed
by
same
letter(s)
do
not
differ
significantly
at
the
5%
level
of
probability
as
calculated
by
Duncan's
multiple
-range
test.
Apparently
contaminated
with
Bacillus
sphaericus/
SS11-1.
346
DAVIDSON
ET
AL.
TABLE
2
AREA
OF
SEALED
BROOD
IN
HONEY
BEE
COLONIES
FED
BACTERIAL
CULTURES
AT
A
CONCENTRATION
OF
I
PART
CULTURE
TO
100
PARTS
20%
SUCROSE
(10
-2
)
Days
after
feeding
started
Bacterial
culture
21
28'
56°
Area
(in.
2
)e
B.
sphaericusISSII-1
79a
104a
254a
B.
sphaericusl
ATCC
7054
94a
94a
157a
20%
Sucrose
control
81a
131a
229a
Measured
immediately
prior
to
removal
from
fl
ight
cages.
6
Measured
after
bees
had
been
free
-flying
for
4
weeks.
Means
in
each
column
followed
by
the
same
letter
do
not
differ
significantly
at
the
5%
level
of
probability
as
calculated
by
Duncan's
multiple
-range
test.
The
colony
without
eggs
was
queenless
,
and
therefore,
a
new
queen
was
introduced.
Subsequently,
this
colony
produced
brood.
The
amount
of
brood
produced
in
all
colo-
nies
was
relatively
low
and
was
probably
due
to
the
"cage
effect".
After
the
colonies
were
moved
from
the
cages
to
an
outside
apiary,
brood
production
returned
to
normal
levels
in
both
the
check
and
treated
colo-
nies.
No
significant
differences
in
brood
production
were
observed
or
measured
in
the
colonies
receiving
the
bacterial
solutions
and
the
20%
sucrose
controls
(Table
2).
The
results
of
this
study
demonstrate
that
B.
sphaericus
strain
SSII-1
affects
neither
the
longevity
of
newly
emerged
adult
bees,
nor
the
brood
production
of
colonies
fed
cul-
tures
of
the
bacterium.
ACKNOWLEDGMENTS
The
authors
thank
Sue
Martynowski
for
her
techni-
cal
assistance.
We
thank
the
Division
of
Agriculture
and
the
College
of
Engineering,
Arizona
State
Uni-
versity,
for
the
use
of
laboratory
space
and
equip-
ment.
This
research
was
supported
in
part
by
National
Science
Foundation
Grant
No.
BMS
72-01954
A01,
and
in
part
by
a
grant
from
Abbott
Laboratories,
North
Chicago,
Illinois.
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