BIOLOGICAL AND MICROBIAL CONTROL

Benzylideneacetone, an Immunosuppressant, Enhances Virulence of

Bacillus thuringiensis Against Beet Armyworm
(Lepidoptera: Noctuidae)
BOWON KWON AND YONGGYUN KIM1
Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea

J. Econ. Entomol. 101(1): 36Ð41 (2008)

ABSTRACT Benzylideneacetone (BZA) is a metabolite of gram-negative entomopathogenic bac-
terium Xenorhabdus nematophila, and it acts as an enzyme inhibitor against phospholipase A2 (PLA2).
PLA2 catalyzes a committed biosynthetic step of eicosanoids, which mediate insect immune reactions
to infection by microbial pathogens. This study tested a hypothesis that a putative immunosuppressive
activity of BZA may enhance virulence of Bacillus thuringiensis against the Þfth instars of Spodoptera

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exigua (Hübner) (Lepidoptera: Noctuidae). In in vitro conditions, BZA signiÞcantly inhibited he-
mocyte microaggregation induced by B. thuringiensis and impaired hemocyte-spreading behavior of
S. exigua in a dose-dependent manner. Oral administration of BZA gave similar immunosuppressive
effect on the hemocytes of the Þfth instars. Although BZA itself did not possess any insecticidal activity
on oral administration, when BZA was treated in a mixture with a low dose of B. thuringiensis spp.
aizawai to Þfth instars, the bacterial virulence was signiÞcantly enhanced. BZA also enhanced
virulence of B. thuringiensis spp. kurstaki, which alone was of limited effectiveness against S. exigua.
This study suggests that an immunosuppression by BZA is positively linked to potentiation of B.
thuringiensis.

KEY WORDS benzylideneacetone, Spodoptera exigua, microaggregation, Bacillus thuringiensis, im-

munosuppression

Eicosanoids are a chemical group encompassing all
biologically active, oxygenated metabolites of arachi-
donic acid and two other C20 polyunsaturated fatty
acids (Corey et al. 1980, Stanley 2005). They are im-
plicated in mediating insect immune reactions in re-
sponse to various microbial pathogens (Stanley 2006).
Cellular immune reactions of hemocyte microaggre-
gation, phagocytosis, and nodulation are mediated by
eicosanoids (Stanley and Miller 2006). Humoral im-
mune reactions, including phenoloxidase activation
and microbial peptide production, also are induced by
eicosanoids (Mandato et al. 1997, Morishima et al.
1997).
Phospholipase A2 (PLA2) performs the committed
catalytic step of eicosanoid biosynthesis (Six and Den-
nis 2000). Challenge with bacteria induces production
of eicosanoids by stimulating PLA2 activity in
Manduca sexta (L.) (Tunaz et al. 2003). Inhibition of
PLA2 signiÞcantly impairs immune reactions, includ-
ing hemocyte spreading (Miller 2005), phagocytosis
(Shrestha and Kim 2007), nodulation (Miller et al.
1996), and antimicrobial peptide production (Yajima
et al. 2003). An entomopathogenic bacterium, Xenorh-
abdus nematophila (Poinar and Thomas), induces a
1 Corresponding author, e-mail: hosanna@andong.ac.kr.
fatal septicemia by speciÞcally inhibiting PLA2 (Park
and Kim 2000, 2003).
Bacillus thuringiensis Berliner (Bt) is a gram-posi-
tive bacterium that forms endospores containing ␦-en-
dotoxins, which target insect midgut epithelium (Jen-
kins and Dean 2000). Despite its high potency and
environmental friendly merits, wide application of Bt
has been limited by its narrow spectrum and devel-
opment of resistance. The beet armyworm, Spodoptera
exigua (Hübner) (Lepidoptera: Noctuidae) is a lepi-
dopteran pest causing serious damage to major vege-
table crops, and it cannot be adequately controlled by
most commercial pesticides because of its wide range
of resistance (Brewer and Trumble 1989, Van Laecke
and Degheele 1991). Its decrease in the pesticide
susceptibility becomes more serious especially when
it develops into late instars (Kim et al. 1998).
Bt tolerance is associated with loss or modiÞcation
of Bt receptors (Gahan et al. 2001, Ferré and Van Rie
2002), altered proteolysis of Bt toxin(s) (Oppert et al.
1997), and recovery of damaged midgut epithelial cells
(Forcada et al. 1999). More recently, the insensitivity
to Bt has been explained in terms of immune reactions
by demonstrating that preexposure to low Bt concen-
tration elevated Bt tolerance and that hemolymph
melanization was highly correlated with Bt tolerance
(Rahman et al. 2004). This causal link between Bt

0022-0493/08/0036Ð0041$04.00/0 䉷 2008 Entomological Society of America

February 2008 KWON AND KIM: BZA ENHANCES VIRULENCE OF B. thuriengensis 37

tolerance and insect immune reactions led us to pose 100

a a
a hypothesis that any PLA2 inhibitor can induce im- a BZA
munosuppression, which then increases Bt suscepti- a

Hemocyte-spreading (%)
80 b
bility of S. exigua at late instars. To test our hypothesis,
we used benzylideneacetone (BZA), a metabolite of
X. nematophila, inhibiting PLA2 of S. exigua (Shrestha 60
and Kim 2007) to increase Bt virulence.
c
40
Materials and Methods c c
Insects and Bt. The S. exigua used in this study 20
originated from a Þeld population infesting Welsh
onion, Allium fistulosum L., in Andong, Korea. The
0
larvae were reared on artiÞcial diet (Gho et al. 1990)
100
at 25⬚C, and the adults were fed 10% sucrose. B. thu- a a
ringiensis ssp. aizawai GB413 (Bta, GreenBioTech, a
a DEX

Hemocyte-spreading (%)
Chungju, Korea) was donated. Their active ingredient 80
b
was 5.27 ⫻ 103 colony-forming units (cfu)/␮g of for-
mulation, even though it consisted of the bacterial
60
spore and endotoxin. A commercial product of B.

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thuringiensis ssp. kurstaki (Btk, Thuricide, Misung Inc.,
Korea) was purchased and used. Its active ingredient
was 3.0 ⫻ 107 cfu/␮g, although it also contained both
bacterial spore and endotoxin.
Chemicals. Two speciÞc PLA2 inhibitors of dexa-
methasone (DEX) [(11␤,16␣)-9-ßuoro-11,17,21-
trihydroxy-16-methylpregna-1,4-dione] and BZA
[trans-4-phenyl-3-buten-2-one] were purchased from
Sigma (St. Louis, MO, USA). Anticoagulant buffer
(ACB) was prepared by adding 8 mg of L-cysteine-
HCl (Sigma) to 5 ml of Tris-buffered saline (50 mM
Tris-HCl, 100 mM dextrose, 5 mM KCl, 2.5 mM MgCl2,
and 50 mM NaCl, adjusted at pH 7.5).
Hemolymph Collection. Final instars of S. exigua
were surface-sterilized using cotton swab soaked in
70% ethanol. After cutting the proleg with a pair of
scissors, the exuding hemolymph sample was col-
lected in 10⫻ volume of ice-cold ACB and gently
shaken to facilitate thorough mixing of hemolymph
and ACB.
Hemocyte-Spreading Assay. Hemocyte monolayers
(50 ␮l each) were prepared on glass slides with 49 ␮l
of hemocyte suspension and 1 ␮l of test solution (or
solvent for control), and they were left in moist cham-
ber at 25⬚C for 45 min in dark. In in vitro assays, seven
serial doses of BZA or DEX were prepared in dimethyl
sulfoxide and tested at Þnal doses from 5 ⫻ 10⫺9 to 5 ⫻
10⫺3 M. The spread hemocytes on the glass slides were
observed at 40⫻ under a BX41 phase contrast micro-
scope (Olympus, Tokyo, Japan) by counting 100 he-
mocytes from a randomly chosen Þeld of view. Each
treatment was replicated three times. The number of
hemocytes attached and spread on the glass surface
were determined as described by Madanagopal and
Kim (2006).
Hemocyte Microaggregation Assay. Hemocytes
were collected from 1 ml of hemolymph in ACB by
centrifugation at 800 ⫻ g for 5 min and then sus-
pended in 200 ␮l of TC100 cell culture medium (JBI,
Daegu, Korea). Each treatment (50 ␮l) consisted of
2 ␮l of test chemical or bacterial solution and 48 ␮l
of the hemocyte suspension. Treatments consisted
40 c
d
20 d
0
-9 -8 -7 -6 -5 -4 -3
0 10 10 10 10 x10 10 10
5x 5x 5x 5x 5 5x 5x
Inhibitor concentration (M)
Fig. 1. Effect of two PLA2 inhibitors, BZA and DEX, on
hemocyte-spreading behavior of the Þfth instars of S. exigua.
Each treatment was replicated three times. Different letters
on the standard deviation bars indicate signiÞcant difference
among means at P ⬍ 0.05 (LSD test).
of 1) control solvent, 2) 4 ppm Bta suspension, 3) 10
␮M BZA, and 4) a mixture of 4 ppm Bta and 10 ␮M
BZA. After 10-min incubation of each treated mix-
ture in 25⬚C, 10 ␮l of each reaction mixture was
loaded onto a hemocytometer (Superior, Bad Mer-
gentheim, Germany) and observed under the phase
contrast microscope. Hemocyte microaggregation
was scored positive when the aggregate was bigger
than a single square (2,500 ␮m2) of the hemocy-
tometer used for counting white blood cells. Inten-
sity of hemocyte microaggregation represented the
number of the squares covered by the aggregates.
Each treatment was replicated three times.
Oral Administration of BZA. An artiÞcial diet (⬇1
cm3) was soaked in 250 ␮M BZA for 10 min and used
to individually feed the Þfth instars of S. exigua for 48 h.
As a control, the diet was soaked into 0.05% ethanol
and used to feed the larvae. Then, hemolymph of the
treated larvae was collected in ACB as described
above. Each replication consisted of a pooled hemo-
lymph of Þve individuals, and it was replicated three
times. Each 50 ␮l of the pooled hemolymph was used
for hemocyte-spreading assay as described above.
Also, the pooled hemolymph was used to collect he-
mocytes, which were assayed for the hemocyte mi-
38 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 101, no. 1

(A)
&RQ

%=
&21 %W$

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%= %W%=$
(B)
Intensity of hemocyte microaggregation

25

a
20

15

10
b
5 b
b

0
CON Bt BZA Bt+BZA
Fig. 2. Effect of BZA on hemocyte microaggregation of the Þfth instars of S. exigua. (A) Photos (50⫻, BX41 phase contrast)
showing hemocyte microaggregation in response to control solvent (CON), 4 ppm B. thuringiensis spp. aizawai (Bt, 5.27 ⫻
103 cfu/␮g) suspension, 10 ␮M BZA or a mixture of 4 ppm Bt and 10 ␮M BZA (Bt ⫹ BZA). (B) Intensity of hemocyte
microaggregations in response to the treatments, in which the intensity was measured by counting squares of hemocytometer
covered by the hemocyte aggregates. Each treatment was replicated three times. Different letters on the standard deviation
bars indicate signiÞcant difference among means at P ⬍ 0.05 (LSD test).

croaggregation by incubating with the heat-killed
Escherichia coli.
Bioassay. All virulence bioassays were conducted by
soaking a small piece of artiÞcial diet (⬇1 cm3) in the
predetermined concentrations of BZA or bacterial
suspension for 5 min. Each Bt treatment represented
Bta (100 ppm) or Btk (1,000 ppm), respectively. Mix-
ture treatment in each Bt strain was Bta (100 ppm) or
Btk (1,000 ppm) with 250 ␮M BZA. As control, the diet
was soaked into 0.05% ethanol. After drying under
dark condition for ⬇10 min, the treated diet was given
to the Þfth instars. Virulence was measured by mor-
tality at 4 d after the bacterial treatment. Larvae were
considered dead when they did not move when prod-
ded with a blunt probe. Each measurement was rep-
licated independently three times with 10 larvae per
replication.
Statistical Analysis. Means and variances of control
and experimental treatments were analyzed with a
one-way analysis of variance (ANOVA) by using
PROC GLM of SAS (SAS Institute 1989). Percent data
were normalized by arsine transformation. ANOVAs
were followed by least signiÞcant difference (LSD)
(P ⫽ 0.05) for mean separation.
February 2008
100
a a
Hemocyte-spreading (%)
80
60
40
20
0 Control
Intensity of hemocyte microaggregation
50 a
40
30
20
10
0 Control
Control
Oral administration for 48 h
Fig. 3. Effect of oral administration of BZA on hemocyte-
spreading and hemocyte aggregation reactions of the Þfth
instars of S. exigua. The treated larvae were fed for 48 h with
artiÞcial diet soaked in 250 ␮M BZA or 0.05% ethanol (con-
trol). Hemolymph from the treated larvae was collected and
used for the cellular immune reactions described in Materials
and Methods. Each treatment was replicated three times.
Different letters on the standard deviation bars indicate
signiÞcant difference among means at P ⬍ 0.05 (LSD test).
Results
Hemocytes of S. exigua larvae could exhibit spread-
ing behavior on glass plate. However, this hemocyte-
spreading behavior was signiÞcantly inhibited by add-
ing BZA (F ⫽ 42.73; df ⫽ 7, 16; P ⬍ 0.0001) (Fig. 1).
DEX, a well-known speciÞc inhibitor to PLA2, also
signiÞcantly inhibited the hemocyte-spreading behav-
ior (F ⫽ 186.32; df ⫽ 7, 16; P ⬍ 0.0001). Both inhibitors
showed their activities in dose-dependent manners at
micromolar range.
The effect of BZA was further analyzed on hemo-
cyte microaggregation behavior of S. exigua larvae
(Fig. 2). Bt induced the hemocyte microaggregation
shown in aggregates, which covered several hemocy-
tometer squares (Fig. 2A). However, when Bt and
BZA were treated together, the formation of hemo-
cyte microaggregation was signiÞcantly impaired (F ⫽
26.26; df ⫽ 3, 8; P ⫽ 0.0002) (Fig. 2B).
When BZA was orally administered, it showed a
signiÞcant inhibition (t ⫽ 22.45, df ⫽ 4, P ⬍ 0.0001) on
hemocyte-spreading behavior (Fig. 3A). The similar
signiÞcant inhibition (t ⫽ 3.83, df ⫽ 4, P ⫽ 0.0186) was
KWON AND KIM: BZA ENHANCES VIRULENCE OF B. thuriengensis 39
(A)
70
60
50
Mortality (%)
40 b
30
b
b 20 b
10
0
Bta BZA Bta+BZA
(B) 100
a
80
Mortality (%)
60
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40
b b
b
b 20
0
Btk BZA Btk+BZA
BZA
Fig. 4. Enhancement of B. thuringiensis virulence in a
mixture with BZA against the Þfth instars of S. exigua. (A)
BZA and Bta (5.27 ⫻ 103 cfu/␮g) was prepared in 250 ␮M and
100 ppm, respectively, and orally administered in single or
mixture. (B) BZA and Btk (3.0 ⫻ 1010 cfu/␮g) was prepared
in 250 ␮M and 1,000 ppm, respectively, and orally adminis-
tered in single or mixture. Virulence was measured by mor-
tality at 4 d after the bacterial treatment. Each measurement
was replicated independently three times with 10 larvae per
replication. Different letters on the standard deviation bars
indicate signiÞcant difference among means at P ⬍ 0.05 (LSD
test).
shown in the analysis of hemocyte microaggregation
(Fig. 3B).
The inhibitory actions of BZA on immune reactions
and its per os effect led us to hypothesize that BZA
could enhance the Bt efÞcacy against S. exigua larvae
(Fig. 4). At 100 ppm, Bta did not give any signiÞcant
mortality against the Þfth instars of S. exigua (Fig. 4A),
although the Bt strain could give signiÞcant virulence
in higher doses (Jung and Kim 2006). BZA was treated
at 250 ␮M, at which any signiÞcant mortality was not
observed. But, there was a signiÞcant mortality in-
crease (F ⫽ 20.32; df ⫽ 3, 8; P ⫽ 0.0004) when the
mixture of Bta and BZA was orally administered.
When Btk was applied at 1,000 ppm, no signiÞcant
mortality was observed. However, it was treated with
250 ␮M BZA, the virulence was signiÞcantly enhanced
(F ⫽ 32.75; df ⫽ 3, 8; P ⬍ 0.0001) (Fig. 4B).
Discussion
Immune responses of target insects may increase
the efÞcacy of microbial pesticides. Rahman et al.
40 JOURNAL OF ECONOMIC ENTOMOLOGY
(2004) advocated the importance of immune re-
sponses in the Bt tolerance by the fact that preexpo-
sure to low Bt concentration elevated Bt tolerance and
that hemolymph melanization (an immune response)
was highly correlated with the development of Bt
tolerance. More recently, Kwon and Kim (2007) dem-
onstrated that pyriproxyfen, a juvenile hormone ag-
onist, inhibited cellular immune reactions and en-
hanced Bt susceptibility of Plutella xylostella (L.).
Thus, inhibition of immune response may increase Bt
virulence. Our study supports this hypothesis in sev-
eral points. First, BZA showed its immunosuppressive
effect by inhibiting hemocyte-spreading and micro-
aggregation behaviors that were induced by microbial
infection. Second, orally administered BZA also im-
paired the hemocyte immune reactions. This suggests
that the orally fed BZA may enter hemocoel to inhibit
hemocyte behavior, resulting to an immunosuppres-
sive state. Finally, mixture treatment of Bt and BZA
signiÞcantly enhanced Bt virulence. It was interesting
that BZA also signiÞcantly enhanced Btk virulence to
the Þfth instar larvae of S. exigua. Btk Cry1A(c)-type
␦-endotoxin, which is highly toxic to many lepidop-
teran pests, was not very efÞcacious against S. exigua
except in its early larval stages (Lastra et al. 1995). A
previous study showed that BZA also inhibited hemo-
cyte phagocytosis in S. exigua (Shrestha and Kim
2007). BZA is a metabolite of X. nematophila and acts
like PLA2 inhibitor (Ji et al. 2004, Shrestha and Kim
2007) because any inhibitory effect of BZA could be
reversed by addition of arachidonic acid, a catalytic
product of PLA2. PLA2 is a common pathogenic mo-
lecular target of two entomopathogenic bacteria, Pho-
torhabdus and Xenorhabdus (Kim et al. 2005). We
infer from these results that eicosanoids mediate im-
mune response to Bt infection.
A functional link between immune reactions and Bt
tolerance still remains speculative. As proposed pre-
viously (Rahman et al. 2004), humoral immune factors
may be secreted into gut lumen and interact with Bt
toxin to cause its inactivation or bind to Bt receptor to
prevent fatal toxin binding. In this study, BZA could
inhibit immune reactions of the Þfth instars of S. exigua
even in its oral administration. In this immunosup-
pressive state, S. exigua larvae may not express effec-
tive defense against Bt, leading to signiÞcant enhance-
ment of the bacterial virulence. The increase of Btk
virulence at a mixture with BZA suggests that immune
capacity of target insects may contribute to determine
Bt host range. Alternatively, the enhancement of Bt
virulence might be induced by indirect pathogenic
effect. A previous study demonstrated that Bt infec-
tion to S. exigua midgut could form pores, through
which materials including potential pathogens in the
gut lumen moved in both directions between gut lu-
men and hemocoel (Jung and Kim 2006). Thus, S.
exigua treated with both Bt and BZA in this study must
have suffered from gut microbes that may enter hemo-
coel through pores produced by Bt infection, which
may easily induce septicemia in immunosuppressive
condition triggered by BZA.
Vol. 101, no. 1
In summary, this study demonstrates an effect of
BZA on the enhancement of Bt virulence by inducing
immunosuppression and suggests that PLA2 inhibitors
or eicosanoid biosynthesis inhibitors can be exploited
to enhance Bt virulence. BZA has been regarded as a
safe chemical because it is used for an industrial ma-
terial for synthesis of chemicals and drugs, and for a
ßavoring additive for cosmetics, soaps, detergents, cig-
arettes, and some foods (Kitamura et al. 1999). This
study proposes a mixture treatment of Bt with BZA or
other eicosanoid-biosynthesis inhibitors to effectively
control Bt-tolerant insect pest population.
Acknowledgments
We appreciate Youngim Song for encouragement and
supplying materials for this research. This study was funded
by Korea Institute of Industrial Technology Evaluation and
Planning (to Y.K.). B.K. was supported by the Second Stage
BK21 program of Ministry of Education and Human Re-
Downloaded from http://jee.oxfordjournals.org/ by guest on June 7, 2016
sources Development.
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Received 23 June 2007; accepted 19 September 2007.