Botanical Studies (2007) 48: 273-281.
*
Corresponding author: E-mail: mbycchao@gate.sinica.edu.
tw; Phone: 886-02-2788-2697.
INTRODUCTION
The baculovirus has long been used as a useful
microbial agent for the control of insect pests. However,
infected insects can still feed on crops during the
incubation period, resulting in significant economic loss.
Therefore, if a toxin gene sequence can be inserted into the
genome of baculoviruses and then expressed by promoters
which can drive the insect-specific toxin earlier and/or
stronger, the insecticidal efficacy can be well accelerated.
Recombinant viruses containing scorpion toxin or mite
toxin can paralyze infected larvae and prevent them from
further damaging crops. These toxin genes can not only
make infected larvae stop eating and prevent the loss of
crops, they also cause the infected larvae to die earlier,
hence strengthening the effect of pest management
(Stewart et al., 1991; Tomalski and Miller 1991; Hoover
et al., 1995; Hughes et al., 1997; Hernandez-Crespo et al.,
1999).
An insect specific neurotoxin from Leiurus
quinquestriatus hebraeus, LqhIT
2
, has effective toxicity
and shortened time to paralysis (Zlotkin et al., 1993;
Benkhalifa et al., 1997; Prikhod¡¦ko et al., 1998; Gershburg
et al., 1998; Regev et al., 2003; van Beek et al., 2003).
However, the key factors that determine the expression of
foreign genes and lethality of neurotoxins are the timing
and intensity of promoter expression (Lu et al., 1996;
Jarvis et al., 1996; Gershburg et al., 1998; van Beek et al.,
2003; Tuan et al., 2005). Previously, the toxin LqhIT
2
was
expressed by early promoters, including CMV minimal
(Tuan et al., 2005), pag90 (Jinn et al., 2006) and p6.9
BIOChemISTRy
high level production of polyhedra in a scorpion toxin-
containing recombinant baculovirus for better control of
insect pests
Shu-Jen TUAN
1,2,3
, Roger F. HOU
3
, Chi-Fen LEE
4
, and Yu-Chan CHAO
2,5,
*
1
Residue Control Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Wufeng, Taichung
413, Taiwan
2
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan
3
Department of Entomology, National Chung Hsing University, Taichung 402, Taiwan
4
National Pingtung University of Science and Technology, Graduate Institute of Biotechnology, Neipu, Pingtung 912,
Taiwan
5
Institute of Life Sciences, College of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan
(Received September 18, 2006; Accepted January 5, 2007)
ABSTRACT.
A sufficient occlusion body yield by a scorpion toxin-containing baculovirus is important to the
success of a pest management program. In this study, recombinant baculoviruses, AcMNPV, vAPcmIT
2
, by
which the scorpion toxin (LqhIT
2
) is driven by an early phase promoter (p-PCm); and another recombinant
baculovirus vAP10IT
2
, by which LqhIT
2
is driven by a very late p10 promoter, were tested for the efficiency
of their polyhedral production. In Sf21 cells, the yield of polyhedra by vAPcmIT
2
is significantly better than
that of vAP10IT
2
. Although in Trichoplusia ni (Hubner) and Spodoptera exigua (Fabricius) the polyhedra
yields by vAPcmIT
2
-infected larvae were not as good as those of the wild type virus, they were about tenfold
higher than those produced by vAP10IT
2
-infected larvae. To test the insecticidal activity of these recombinant
baculoviruses, vAPcmIT
2
and vAP10IT
2
were applied against two major pesticide-resistant vegetable pests,
Plutella xylostella (Linnaeus) and S. exigua. Our results demonstrated a significant shortening of the lethal
time (LT
10
and LT
50
) compared to those larvae infected with wild type AcMNPV. In field trials, larvae of S.
exigua infected with the toxin-recombinant viruses provided more than 90% control efficacy and resulted in
a 58.7~67.4% reduction in leaf area consumed compared to wild type AcMNPV. Based on the efficacy of
polyhedral production and crop protection, the superiority of vAPcmIT
2
over both vAP10IT
2
and wild type
AcMNPV renders it a better candidate to serve as a useful biopesticide.
Keywords: Autographa californica nucleopolyhedrovirus; AcMNPV; Leiurus quinquestriatus hebraeus;
Insecticidal efficacy; Neurotoxin; Plutella xylostella; Polyhedral production; Recombinant virus; Spodoptera
exigua; Trichoplusia ni.
pg_0002
274
Botanical Studies, Vol. 48, 2007
(Fujita et al., 2006) promoters. In these experiments,
the early promoters were shown to be more efficient
insecticides than very late promoters (p10, vAP10IT
2
).
Mass production of baculovirus is a bottleneck in the
application of microbial insecticides. The yields can vary
with pathogenicity of the virus, inoculum concentration,
larval stage, body weight, neurotoxin expressed, and the
timing and intensity by which the promoter is expressed
(Shapiro, 1986; Bonning et al., 1995; Tuan et al., 1995;
Ignoffo and Garcia, 1996; 1997; Burden et al., 2000;
Harrison and Bonning, 2000). Thus, if an engineered
baculovirus is to become an effective biological control
agent, its proper production is necessary.
In previously studies, p10, the very late promoter,
was used most often to drive heterologous proteins
in recombinant baculoviruses because of its powerful
expression capability (Stewart et al., 1991; Hughes et al.,
1997; Burden et al., 2000; Regev et al., 2003). However,
the use of Ro10AaIT and Ro10LqhIT
2
, AcMNPV p10
promoter resulted in a reduction of polyhedral production
in vitro (Harrison and Bonning, 2000). The reduced yield
of polyhedra can be due to competition among p10 and
polh promoters, both strong very late promoters, for
transcriptional and/or translation factors (Chaabihi et al.,
1993; Volkman et al., 1996). Presumably such competition
would be resolved if the expression time of the toxin and
polyhedra genes could be separated.
In this report, we compared the effect of the early
p-PCm and very late p10 promoters on their production
of polyhedra. The p-PCm promoter contains the human
cytomegalovirus minimal (CMVm) promoter ligated in cis
with the polyhedrin upstream (pu) sequence. This results in
a high-level of expression for the foreign genes at the early
infection stage of the baculovirus (Wu et al., 2000; Lo et
al., 2002). Different insect species and cell lines were used
to produce polyhedra of recombinant viruses that express
the toxin protein LqhIT
2
using p10 and p-PCm promoters.
The insecticidal efficacy and the management potential
of the recombinant viruses for different insects using p10
and p-PCm promoter were also evaluated. Importantly,
in the expression of toxin gene using CMVm and p10
promoters in different recombinant viruses, the former was
found to produce more polyhedra than the latter. These
results will be useful for polyhedral production in the
practical application of toxin gene-containing recombinant
baculoviruses for pest control in the fields.
mATeRIALS AND meThODS
Cell lines and viruses
Spodoptera frugiperda IPLB-Sf21AE (Sf21, Vaughn
et al., 1977) cells were maintained and propagated in
a modified TNM-FH medium containing 8% heat-
inactivated fetal bovine serum at 26¢XC (Lee et al., 1998;
Lin et al., 1999). The C6 strain of wild-type AcMNPV
(C6-AcMNPV), and two toxin-gene-containing viruses,
vAP10IT
2
, and vAPcmIT
2
(Tuan et al., 2005) were
propagated in the Sf21 cell line under the conditions
mentioned above. On day 7 post infection (p.i.), the
polyhedra were collected and quantified (O¡¦Reilly et
al., 1992; Tuan et al., 2005). vAP10IT
2
and vAPcmIT
2
were recombinant AcMNPVs which contained LqhIT
2
expressed by two temporal promoters, the very late p10
and the early p-PCm, respectively (Tuan et al., 2005).
Insects
Larvae of the diamondback moth P. xylostella and the
cabbage looper T. ni were collected from Shi-hu County,
Taiwan and reared on an artificial diet (Tuan et al., 1997).
Colonies were maintained at 22¡Ó1¢XC with 70¡Ó5% relative
humidity (RH) and a 12L:12D photoperiod. Larvae of
the beet armyworm S. exigua (Hubner) were collected
from Shi-hu County, Taiwan and reared on an artificial
diet (Tuan et al., 1997). Colonies were maintained at
25¡Ó1¢XC under the above-mentioned conditions. All
insects tested were 2
nd
, 3
rd
, o r 4
th
instar larvae after
rearing for three generations. Sanitation procedures for
maintaining colonies were applied as a precaution against
contamination with microorganisms (Tuan et al., 2005).
The insects in all experiments were observed daily by
smears under a phase-contrast microscope and checked
for evidence of baculovirus infection. For bioassays,
all molting larvae of the same age were selected and
enclosed individually in a 30-well plate overnight. Next
morning, the newly molted larvae were starved for 8 h to
synchronize larval growth.
Quantification of polyhedra
Sf21 cells were seeded at 10
6
cells/well into 6-well
plates (Falcon
.
) in TNM-FH medium and allowed to
attach for 2 h at 26¢XC. Following removal of the spent
medium, 0.5 ml C6-AcMNPV, vAP10IT
2
, and vAPcmIT
2
were added to each well at m.o.i.=1 in triplicate. The
inoculated cells were re-incubated at 26¢XC for 8 days.
Visual examination of the infection processes was
performed daily until all samples were harvested.
Collection and quantification of polyhedra was carried
out on day 5, 6, 7, and 8 p.i. (O¡¦Reilly et al., 1992; Tuan
et al., 2005). Polyhedra were isolated from Sf21 cells
infected with C6-AcMNPV, vAP10IT
2
, and vAPcmIT
2
,
and diluted to 10
5
, 10
6
, 10
7
, and 10
8
PIBs/ml. Third-instar
S. exigua and 4
th
-instar T. ni were fed on an artificial diet
contaminated with polyhedral suspensions at the doses
of 7.8, 78, 780, and 7800 PIBs/mm
3
diet, and reared
individually in 30-well plastic plates (Tuan et al., 1997).
Inoculated larvae were collected on day 4, 5, and 6 p.i.
The polyhedra were quantified according to the method
mentioned above. Thirty larvae in three replicates were
inoculated in each treatment. All treated larvae were
weighed on day 5 p.i.
measurement of lethal times
Third-instar larvae of P. xylostella and S. exigua were
fed on a diet contaminated with C6-AcMNPV, vAP10IT
2
,
pg_0003
TUAN et al. ¡X High level polyhedral production of a baculovirus
275
and vAPcmIT
2
at a dose of 780 PIBs/mm
3
. Three days
post-inoculation, all assayed larvae were fed a virus-free
diet. The initial lethal time (10% lethal time, LT
10
) and
median lethal time (LT
50
) were determined. Ninety larvae
in three replicates were inoculated in each treatment. The
larvae were stimulated with a hairbrush and scored as
responders if they were paralyzed or dead (Tuan et al.,
2005).
Field trial
Forty newly molted 2
nd
-instar larvae of S. exigua were
transferred to potted cabbage ca. 60 cm
2
in size with 12~14
leaves. Each pot was confined in a fine-mesh nylon cage
on the trial farm of Taiwan Agricultural Chemicals and
Toxic Substances Research Institute, Wufeng, Taichung,
Taiwan. The cabbages were treated individually with C6-
AcMNPV, vAP10IT
2
, vAPcmIT
2
at 10
7
PIBs/ml, and
sterile water (as a control) using a handheld sprayer. The
spraying volume was 1 ml per leaf. Tween-20 (0.05%) was
added to all viral suspensions and controls. Three potted
cabbages were tested for each treatment. The insects
were checked twice daily to record signs and symptoms
of disease and death using the protocol referred to by
Tuan et al. (2005). Two applications were conducted at an
interval of 7 days. Survival rate, average body weight, and
leaf area eaten were calculated on day 7 after the second
application of viral suspensions as they were after the first
application. All leaves were xeroxed and scanned with
a leaf area meter (Model Li-3100 Area Meter, Li-Cor,
USA), the area eaten by larvae was calculated as referred
to by Tuan et al. (2005). The survival rate was equal to the
number of survival larvae of treated group divide by the
number of tested larvae, and the value was then multiplied
by 100%. The control efficacy was equal to the leaf area
eaten by the larvae of the virus-treated set divided by the
leaf area eaten by larvae of control, and the value was then
multiplied by 100%.
Statistical analysis
All of the bioassays, polyhedral production assays,
and field trials were performed at least thrice. Mortalities
were corrected with Abbott¡¦s formula, and LTs values
were calculated by the probit analysis (Finney, 1971). The
polyhedral production and field trial values for each virus
were averaged and analyzed by one-way ANOVA followed
by the Least Significant Difference Test (Steel and Torrie,
1980).
ReSULTS
Comparison of polyhedral production in vitro
On day 7 p.i., all the cells infected with C6-AcMNPV
(Figure 1A) became extremely swollen and filled with
dozens of polyhedra of regular size. Moreover, some cells
were lyzed to release polyhedra. However, significantly
fewer polyhedra were formed in the vAP10IT
2
-infected
cells (Figure 1B) than those produced by the vAPcmIT
2
-
infected cells (Figure 1C). The production curves of
all infected cells continued to rise until day 7 p.i., then
plateaued on day 8 p.i. (Figure 2). Sf21 cells infected
with C6-AcMNPV, vAP10IT
2
, and vAPcmIT
2
viruses had
polyhedral production of 3.48¡Ñ10
7
, 1.33¡Ñ10
7
, and 3.93
¡Ñ10
7
PIBs/10
6
cells on day 7 p.i., respectively. Among
these viruses, vAP10IT
2
, the recombinant virus expressing
LqhIT
2
under the very late p10 promoter produced the
Figure 1. The micrographs of the occlusion bodies produced in
the Sf21 cells infected with wild type AcMNPV (Panel A), and
toxin-expressing recombinant viruses, vAP10IT
2
(Panel B) and
vAPcmIT
2
(Panel C), on day 7 p.i.
pg_0004
276
Botanical Studies, Vol. 48, 2007
least polyhedra in all the infection stages. On day 8 p.i.,
compared with wild type C6-AcMNPV, the polyhedra
produced by vAP10IT
2
was reduced by 57% in Sf21 cells.
vAPcmIT
2
-infected Sf21 cells produced 12.7% more
polyhedra than C6-AcMNPV-infected cells although this
difference was not significant based on results of the Least
Significant Difference Test with a 95% confident limit.
The vAPcmIT
2
-infected Sf21 cells produced polyhedra
amounts as high as 2.82-fold that of vAP10IT
2
-infected
Sf21 cells (Figure 2).
Comparison of polyhedral production in vivo
On day 5 p.i., the average weight of T. n i larvae
infected with C6-AcMNPV, vAP10IT
2
, and vAPcmIT
2
was 54.0, 38.8, and 36.4 mg/larva, respectively. All
weighed less than the control group larvae, averaging
108.9 mg/larva. The average weights of larvae infected
with the scorpion toxin-containing viruses were 30~50%
less than those infected with wild type viruses. Among
the larvae infected with toxin gene-containing viruses, no
significant difference emerged between the use of early
and late promoters (Figure 3). On day 6 p.i., the polyhedral
production in vAP10IT
2
and vAPcmIT
2
-infected larvae
was far less than that in the wild type viruses-infected
larvae. The yields of polyhedra in insects with a low
inoculated concentration of C6-AcMNPV, vAP10IT
2
,
and vAPcmIT
2
viruses were 3.79¡Ñ10
8
, 3.50¡Ñ10
7
and 1.33
¡Ñ10
8
PIBs/larva, respectively. These data showed that
the polyhedral productions in the larvae infected with
vAP10IT
2
and vAPcmIT
2
were only 9.2% and 35.1% of
those in larvae infected with the C6-AcMNPV.
The larvae treated with higher concentrations of these
viruses had higher overall polyhedral production than the
larvae treated with lower viral concentrations. After the
inoculation of the C6-AcMNPV, vAP10IT
2
, and vAPcmIT
2
viruses, the average polyhedral production of each
infected larva was 7.91¡Ñ10
8
, 3.86¡Ñ10
7
and 3.58¡Ñ10
8
PIBs/
larva, respectively. These data showed that the polyhedral
productions by vAP10IT
2
and vAPcmIT
2
were 4.1% and
40.3% of that produced by wild type virus
(Figure 4). Even
after raising the inoculum concentration 100 fold, the yield
of polyhedra in S. exigua larvae is still around one-fourth
that of T. n i (Figure 5). Therefore, T. n i is better than S.
exigua for polyhedral production. The average yield of
polyhedra per mg body weight of T. ni larvae infected
with vAPcmIT
2
was around 10 fold that with vAP10IT
2
,
and about 64% of that with wild type virus C6-AcMNPV
(Figure 6).
Determination of the lethal activity of
recombinant viruses
The lethal activity of recombinant viruses was
determined and compared with wild-type AcMNPV
(Table 1). The infection of P. xylostella 3
rd
-instar larvae
by vAPcmIT
2
showed that the LT
10
was 11% and LT
50
was
22% earlier than that of C6-AcMNPV-infected larvae.
However, the acceleration of LTs in S. exigua larvae was
Figure 2. Comparisons of polyhedral production in Sf21 (10
6
)
cells infected with recombinant and wild-type AcMNPVs. The
polyhedra were harvested from 5 to 8 days post inoculation.
Figure 3. Average body weight of Trichoplusia ni larvae after
infection with different viruses. Average body weights (means
¡Ó
SD) of T. ni larvae on day 5 post inoculation with recombinant
virus or wild type AcMNPV at a concentration of 78 PIBs/mm
3
.
Each bar labeled with different letters arise significantly different
at P<0.05, Least Significant Difference.
Figure 4. The polyhedral production in Trichoplusia ni larvae
infected with wild-type AcMNP V or recom binant viruses at
a concentration of 7.8 and 78 P IBs/mm
3
, and then harvested
on day 6 post inoculation. Each bar in the same color labeled
with different letters is significantly different at P<0.05, Least
Significant Difference.
pg_0005
TUAN et al. ¡X High level polyhedral production of a baculovirus
277
much less significant than in P. xylostella. The LT50 of
toxin recombinant virus-infected S. exigua larvae was
reduced by 8% to 13% compared to C6-AcMNPV-infected
larvae. Tests on the larvae of these two lepidopteran
species, which are susceptible to AcMNPV, demonstrated
that expression of scorpion toxin could accelerate the
lethal effect (Table 1).
Field efficacy of recombinant viruses
Beginning from day 3 post application (p.a.), the
2
nd
or 3
rd
instar larvae of S. exigua were found to fall
from the potted cabbages sprayed with vAP10IT
2
and
vAPcmIT
2
. These larvae were paralyzed, but did not
show the symptoms of swelling or liquefying typical of
wild-type AcMNPV-infected larvae. The infected larvae
even remained on cabbage leaves and stopped eating
while the larvae treated with C6-AcMNPV or Tween-20
(0.05% in sterile water) continuously ingested leaves
and gained weight. Seven days after the 2
nd
application
with viruses, the vAPcmIT
2
group had the lowest larval
survival rate at 20.3%, followed by the vAP10IT
2
group at
27.3%. The survival rates of the C6-AcMNPV and control
groups were 35.4% and 100%, respectively. There were
significant differences in survival rates among all groups
(Table 2). However, there was no significant difference
between vAP10IT
2
and vAPcmIT
2
treated groups with
regard to the average weight of surviving larvae or the
leaf area eaten. Both groups had a much lower average
weight than those groups treated with wild type viruses.
In addition, the leaf areas eaten by the larvae sprayed
with vAP10IT
2
and vAPcmIT
2
were 58.7% and 67.1%
smaller than those sprayed with wild type virus. All virus-
treated groups had less feeding activity than the non-
virus treated control (Table 2). These results indicated
that the insecticidal efficacies of the toxin-recombinant
viruses are considerably better than those of the wild type
virus based on the larval survival rate in the field trial, the
average weight of survived larvae, and the leaf area eaten.
vAPcmIT
2
showed significantly better insecticidal efficacy
than vAP10IT
2
with respect to larvae survival rate, but
these two viruses showed no significant differences in
average body weight or leaf area eaten.
DISCUSSION
AcMNPV, a pathogen of more than 30 species of
lepidopteran pests, has developed into a useful microbial
insecticide over the past few decades (Bonning and
Hammock, 1996). However, an infected larvae can
continue to cause economic damage during the incubation
period before its death (Ignoffo, 1973; Granados and
Williams, 1986; Groner, 1986; Smits and Vlak, 1988;
Granados and Williams, 1994; Tuan et al., 1998).
Improvement to the efficiency of AcMNPV as an
F igu re 5. The polyhedral production in Spodopter a exigua
l arva e infe cte d with wil d-type AcMNP V or re com bina nt
viruses at a concentration of 780 and 7,800 PIBs/mm
3
and then
harvested on day 6 post inoculation. Each bar in the same color
labeled with different letters is significantly different at P<0.05,
Least Significant Difference.
Figure 6. The average yield of polyhedra per mg body weight
of Trichoplusia ni larvae infected with wild-type AcMNP V or
recombinant viruses at a concentration of 78 PIBs/mm
3
, and
the n harvested on day 6 post inoculation. Each bar labeled
with different letters are significantly different at P<0.05, Least
Significant Difference.
Table 1. Comparison of lethal time of Spodoptera exigua and
Plutella xylostella 3
rd
instar larvae inoculated with recombinant
or wild type baculoviruses
1, 2, 3
Virus
(10
7
PIBs/ml)
Spodoptera exigua Plutella xylostella
LT
10
(h) LT
50
(h) LT
10
(h) LT
50
(h)
AcMNPV
137.1
b
182.3
b
89.0
b
138.2
b
vAP10IT
2
127.5
a
167.9
a
79.5
a
116.4
a
vAPcmIT
2
123.4
a
168.4
a
78.9
a
107.6
a
1
Ti m e i n ho u rs wh e n l a rv a e w e re pl a c e d o n t he di e t
contaminated with 780 PIBs/mm
3
.
2
LT
10
and LT
50
values were calculated us ing probit analysis
with 95% confident limits.
3
Means within a column labeled with different letters were
significantly different at P<0.05, Least Significant Difference.
pg_0006
278
Botanical Studies, Vol. 48, 2007
insecticide is dependent upon reduction of the incubation
period. Scorpion and spider neurotoxins have been
the most widely used foreign toxin proteins and have
transformed baculoviruses into potent bio-insecticides
(Maeda et al., 1991; McCutchen et al., 1991; Cory et al.,
1994; Hoover et al., 1995; Hughes et al., 1997; Gershburg
et al., 1998; Harrison and Bonning, 2000; Burden et
al., 2000). Leiurus quinquestriatus hebraeus excitatory
and inhibitory neurotoxins (LqhIT
1
and LqhIT
2
) are
both considered promising toxins able to accelerate the
speed of kill of baculoviruses, and they can even work
synergistically to reach a higher effect when coexpressed
(Regev et al., 2003).
In order to successfully engineer these toxin genes
into insect viruses for better insecticidal efficacy, strong
early expression of the toxin gene is critical (Difalco et
al., 1997; Gershburg et al., 1998; Harrison and Bonning,
2000; van Beek et al., 2003). It has been demonstrated
that high level expression of LqhIT
2
and LqhIT
1
i n
recombinant viruses by the strong very late promoters
polh and p10 can improve the effective paralysis time
50% (ET
50
) significantly, and their efficacy is better than
that of the early p35 promoter (Gershburg et al., 1998). In
the expression system of insect cells using recombinant
baculovirus, the p-PCm promoter showed a high level
of foreign protein expression at the early stage (Lo et
al., 2002) and expressed scorpion toxin around 12 h
post infection, approximately 12~18 h earlier than that
observed under the p10 promoter (Tuan et al., 2005). This
is also supported by luciferase expression of the p-PCm
promoter, which resulted in higher luciferase activity than
the p10 promoter at early stages of infection, although the
latter expressed more proteins than the former at the very
late stage (Wu et al., 2000; Lo et al., 2002). It has been
previously reported that only a trace amount of scorpion
toxin is required to cause paralysis and death in larvae
(McCutchen et al., 1991; Gershburg et al., 1998). Thus,
the early expression of toxin by the p-PCm promoter is
still sufficient to lead to early paralysis and larval death.
Our experiments showed that the expression of the
LqhIT
2
by vAP10IT
2
and vAPcmIT
2
accelerated the
insecticidal effect on the larvae of T. ni , P. xylostella,
and S. exigua. The LT
50
of T. ni infected with vAPcmIT
2
was significantly shorter than that of vAP10IT
2
-infected
larvae (Tuan et al., 2005), but in the case of P. xylostella,
and S. exigua larvae, there were no significant differences
between larvae infected with vAPcmIT
2
and vAP10IT
2
.
Previously, both RoMNPV recombinant viruses expressing
AaIT and LqhIT
2
from the p10 promoter produced a lower
quantity of viral occlusions than other recombinant viruses
from the p6.9 promoter. Presumably, the relative position
of the strong p10 promoter in those viruses relative to
the polh promoter may be accountable for the decreased
polyhedrin protein accumulation and occlusion assembly
(Harrison and Bonning, 2000). Studies have shown that
expression from the p10 promoter can have a negative
effect on the expression from the polyhedrin promoter
polh due to a resource competition for protein synthesis
(Roelvink et al., 1992; Chaabihi et al., 1993; Bonning et
al., 1994; Volkman et al., 1996).
Economically sound production of polyhedra is
important for the practical application of toxin-gene
engineered baculoviruses. The scorpion toxin causes the
infected larvae to cease eating in a shorter time relative to
wild type, resulting in a significant reduction in polyhedral
production (Cory et al., 1994; Ignoffo and Garcia, 1996;
Fuxa et al., 1998; Burden et al., 2000). Similarly, when
propagating polyhedra of AcAaIT toxin-recombinant
viruses in the larvae, the yield of polyhedra obtained
was only 20% of that for C6-AcMNPV
(Kunimi et al.,
1996). In our study, the toxin-containing recombinant
viruses caused most infected larvae of S. exigua or T. n i
to stop eating, become paralyzed, or even die early before
swelling. At 5 days p.i., the average weight of T. ni larvae
infected with recombinant viruses was 30% less than that
of the larvae infected with wild type virus. Polyhedral
production in vAP10IT
2
and vAPcmIT
2
infected larvae
were about 96% and 60% less than that observed for wild
type AcMNPV. Bonning et al. (1995) pointed out that
the polyhedral production in cells in vitro is 1.5 times
greater than that in larvae, but neither sets of polyhedra
showed any significant difference in the lethal time to T.
Table 2. Comparison of survival and body weight of Spodoptera exigua 2
nd
instar larvae inoculated with recombinant viruses or
wild type AcMNPV and leaf area eaten
1 , 2
.
Virus
(10
7
PIBs/ml)
Survival rate (%)
Average body weight (mg)
Leaf area eaten (cm
2
)
Mean
SD
Mean
SD
Mean
SD
AcMNPV
35.4
c
4.1
30.4
b
5.3
70.0
b
13.6
vAP10IT
2
27.3
b
3.2
20.1
a
3.2
28.9
a
6.7
vAPcmIT
2
20.3
a
2.4
22.9
a
4.4
22.8
a
3.5
Control
100.0
d
0.0
118.2
c
11.8
337.4
c
33.8
1
All viral suspensions were 10
7
PIBs/ml, adjuvant with Triton
ÆÊ
at 2,000-fold dilution, and data were calculated on day 7 after the 2
nd
application at the interval of 7 days from the 1
st
application on day 0.
2
Means within a column labeled with different letters are significantly different at P<0.05, Least Significant Difference. SD,
standard deviation.
pg_0007
TUAN et al. ¡X High level polyhedral production of a baculovirus
279
ni and Heliothis virescens. In our study, the production of
vAPcmIT
2
in Sf21 cells was similar as that of wild type
virus AcMNPV.
Cory et al. (1994) and Hoover et al. (1995) indicated
that recombinant virus with AaIT toxin paralyzed larvae,
stopped their eating, and even caused them to fall from
the plants, reducing the leaf areas consumed on host
plants. In this study, we found that batches of S. exigua
larvae fall to ground 2~3 days p.a., and vAP10IT
2
could
further reduce the leaf area consumed to 67.4% of that
consumed using the wild type virus. Based on the damage
to area of the leaves, the control efficacy performed by
vAPcmIT
2
reached 93.3% as compared with the control
group. In conclusion, our studies showed that scorpion
toxin driven by the early phase promoter is superior to that
driven by the very late promoter in killing lepidopteran
larvae. More importantly, expression of the toxin gene by
the early phase promoter gives rise to better polyhedral
yields in insect cells than that by the very late promoter. A
higher polyhedral production will be useful for the future
application of this virus in the fields for effective control
of insect pests.
Acknowledgements. This study was supported by
research grants No. NSC 90-2313-B-001-014 from the
National Science Council, ROC and #92S202921 from
Academia Sinica. Thanks are extended to Ms. Hsiu-chao
Lin and Mai-yueh Lin for their technical assistance.
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