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.

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

,

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.

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.

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.

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.

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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