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Development of a recombinant baculovirus expressing a modified juvenile hormone esterase with potential for insect control.

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Archives of Insect Biochemistry and Physiology 30:177-194 (1995)
Development of a Recombinant Baculovirus
Expressing a Modified Juvenile Hormone
Esterase With Potential for Insect Control
B.C. Bonning, K. Hoover, T.F. Booth, S. Duffey, and B.D. Hammock
Departments of Entomology and Environmental Toxicology, University of California,
Davis, California (B.C.B., K.H., S.D., B.D.H.); NEXCInstituteof Virology and Environmental
Microbiology, Oafo~d,UK (TEB.)
Baculovirus insecticides are receiving renewed attention as insect pest control
agents following the development of fast-acting recombinant baculoviruses. Here
we report on the construction and biological activity of a recombinant baculovirus derived from the nuclear polyhedrosis virus of Autograph californica
which expresses a modified form of juvenile hormone esterase (JHE).The serine
at the catalytic site of the JHE has been mutated to a glycine residue so that the
protein does not degrade JH. The recombinant baculovirus expressing this modified form of JHE, named AcJHE-SG, has enhanced activity against lepidopteran
larvae. Lethal times of the recombinant are 20 to 30% lower than for the wild type
virus, and a 66% reduction in feeding damage caused by infected larvae is observed. This result is comparable to the best recombinant baculovirus developed to
date, AcAalT, which expresses an insect-selective scorpion toxin. The potential of
these recombinant viruses for commercialization as insecticides is discussed. Bioassays of AcJHE-SG in conjunction with anti-JH agents indicate that the virus is not
killing by an anti-JH mechanism. Larvae apparently die from contraction-paralysis,
or disruption of the normal sequence of events at the molt. o 1995 Wiley-Liss, Inc.
Key words: juvenile hormone esterase, Autographa californica nuclear polyhedrosis
virus, Heliothis virescens
INTRODUCTION
Baculoviruses are arthropod specific viruses which have long been in use
as insect pest control agents for protection of numerous crop plants (Entwistle
Acknowledgments: We thank B.F. McCutchen for provision of the virus AcAalT and A. Vaughen
for assistance with bioassays. This work was supported by DCB-91-19332 from NSF, 91 -373026186 and 92-37302-7601 from USDA, and 23-696 from the USDA Forest Service. B.C.B. was
supported by a NATO collaborative research grant (CRG 900955) and B.D.H. by the Burroughs
Wellcome Foundation to work with R.D. Possee and T.F. Booth, NERC Institute of Virology and
Environmental Microbiology, Oxford, U.K.
Received October 7, 1994; accepted March 8, 1995.
Address reprint requests to Dr. B.D. Hammock, Departments of Entomology and Environmental
Toxicology, University of California, Davis, CA 9561 6.
B.C. Bonning i s now at Department of Entomology, Iowa State University, Ames, IA 5001 1 .
0 1995 Wiley-Liss, Inc.
178
Bonning et al.
and Evans, 1985). According to environmental conditions, death can occur
anywhere from several days to weeks post infection. A reduction in the lethal time of the virus would enable broader use of the virus for protection of
crops less able to sustain foliar damage without economic loss. The main aim
of this work is to develop such a fast-acting baculovirus for commercial use
as a crop protection agent.
Recent advances in recombinant DNA technology have facilitated genetic
engineering of baculoviruses to reduce the time taken for the virus to kill its
larval host (Bonning and Hammock, 1992).Several recombinant baculoviruses
derived from Autographa californica nuclear polyhedrosis virus (AcNPV) with
reduced lethal times have been constructed (Bonning and Hammock, 1994;
Maeda, 1989; McCutchen et al., 1991; OReilly and Miller, 1989; Stewart et al.,
1991; Tomalski and Miller, 1992). The virus AcAaIT", which expresses an insect-selective scorpion toxin (McCutchen et al., 1991; Stewart et al., 1991), is
widely considered to be the best. AcAaIT kills consistently between 25 and
30% more quickly than the wild type parent virus, and does not suffer from
the stability problems seen for some of the other recombinants.
Juvenile hormone (JH) and juvenile hormone esterase (JHE) are key components in the regulation of development in lepidopteran larvae (Hammock,
1985; Riddiford, 1994).As such they have been exploited for use in pest control with development of the juvenoids (Staal, 1982)and anti-JH agents (Staal,
1986) and insertion of JHE into recombinant baculoviruses (Bonning and Hammock, 1994; Bonning et al., 1992; Hammock et al., 1990). A series of mutant
forms of JHE were made by site-directed mutagenesis for analysis of key
residues involved in catalysis of JH (Ward et al., 1992). JHE is a serine esterase with a Ser201-Hi~446-G1~332
catalytic triad (Hanzlik et al., 1989; Ward et
al., 1992). The recombinant viruses expressing the modified forms of JHE were
tested for insecticidal efficacyby bioassay in Heliothis vivescens and Trickoplusia
ni. The virus AcJHE-SG, which expresses the protein with the catalytic site
serine (Serzol)changed to Gly, was found to be highly insecticidal. The efficacy of the virus against lepidopteran larvae is comparable to that of the
virus AcAaIT (McCutchen et al., 1991; Stewart et al., 1991).
In order to gain insight into the mechanism of action of AcJHE-SG, a series
of bioassays were carried out using viruses in conjunction with various chemical agents designed to interfere with regulation of JH. The anti-JH agent ETB
acts as an anti-JH agent at low doses at the tissue level. ETB may reduce JH
*Abbreviations used: AcNPV C6 = Autograph californica nuclear polyhedrosis virus strain C6;
AcAalT = AcNPV expressing the scorpion toxin Androctonus australis Insect-selective Toxin;
AcUW2(B).JHE,AcJHE = AcNPV expressing wild type JHE; AcJHE-DN = AcNPV expressing JHE
with Asp173 changed to Asn; AcJHE-HK = AcNPV expressing JHE with His446 changed to Lys;
AcJHE-RH = AcNPV expressing JHE with Argd7 changed to His; AcJHE-SG = AcNPV expressing
JHE with Serzol changed to Gly; ANOVA = analysis of variance; BSA = bovine serum albumin;
CL = confidence limits; ECL = enhanced chemi-luminescence; EPPAT = O-ethyl-S phenylphosphoramidothiolate; ETB = ethyl 4-(2-(tert-butyl-carbonyloxy)butoxy)benzoate); FCS = fetal calf serum; FMev = Fluoromevalonolactone, tetrahydro-4-fluoromethyl-4-hydroxy-2H-pyran-2-one; J H
= juvenile hormone; JHE = juvenile hormone esterase; LRD = lethal ratio for dose; LRT = lethal
ratio for time; OTFP = 3-octylthio-1, I , I -trifluoropropan-2-one; PAGE = polyacrylamide gel electrophoresis; PEG = polyethylene glycol; pibs = polyhedron inclusion bodies; rJHBP = recombinant juvenile hormone binding protein; SDS = sodium dodecyl sulphate; TLC = thin layer
chromatography.
Baculovirus Expressing Modified JHE
179
titers by acting on the feedback regulation of JH production by the corpora
allata (Staal, 1986).At high doses ETB acts as a juvenoid (Kiguchi et al., 1984;
Kramer and Staal, 1981; Sparks et al., 1979). FMev is an anti-JH agent which
acts directly on the corpora allata. FMev acts as a competitive inhibitor of
mevalonic acid in conversion of mevalonate to JH (Quistad et al., 1981). The
juvenoids, epofenonane, and fenoxycarb were used in bioassays along with
the JHE inhibitors EPPAT (Sparks et al., 1983) and OTFP (Abdel-Aal and Hammock, 1985). These compounds are very potent inhibitors of JHE but also
inhibit other esterases to varying degrees. Here we report on the action of
AcJHE-SG and demonstrate its potential for insect control.
MATERIALS AND METHODS
Chemical Reagents
The juvenoids fenoxycarb (Dorn et al., 1976) and epofenonane [Ro-10-3108; 1(4'-ethylphenoxy)-6,7-epoxy-3-ethyl-7-methylnonane
(Zurfluh et al., 196811, supplied by U. Schweiter, Hoffman-LaRoche(Neuilly, France), were used in bioassays.
ETB (ZR 2646) was supplied by G. Quistad and G.B. Staal formerly of Zoecon
Corporation (Palo Alto, California). FMev was supplied by D.A. Schooley, formerly of Zoecon Corporation. The JHE inhibitor EPPAT was supplied by T.R.
Fukuto (Universityof California, Riverside) and J. Sanborn (California EPA, Sacramento, California). OTFP was synthesized as described (Hammock et al., 1984)
and purified by vacuum distillation. Further analyses showed it to be a single
spot on normal phase TLC and a single peak on capillary GLC.
Viruses
AcJHE-SG is a recombinant baculovirus which expresses juvenile hormone
esterase with the catalytic serine (Ser201)mutated to Gly. Hence, this modified protein has no catalytic activity against JH (Ward et al., 1992). The viruses AcUW2(B).JHE (AcJHE; Bonning et al., 1992), AcJHE-HK, AcJHE-DN,
and AcJHE-RH (Ward et al., 1992) were employed as recombinant control
viruses. AcJHE-HK expresses JHE with His446changed to Lys; AcJHE-DN expresses JHE with
changed to Asn, and AcJHE-RH expresses JHE with
Argd7changed to His. AcJHE-HK, AcJHE-DN, and AcJHE-RH were constructed in addition to AcJHE-SG for analysis of the catalytic mechanism of
JHE (Ward et al., 1992).None of the JHEs produced by these viruses are highly
active in hydrolyzing JH. All recombinant viruses were derived from the wildtype AcNPV strain C6, and were constructed using the plasmid pAcUW2
(Weyer et al., 1990) such that expression of the modified JHE gene was driven
by a duplicated viral p10 promoter. Expression of recombinant proteins from
these viruses in vitro is similar (Ward et al., 1992). The recombinant virus
AcAaIT, which expresses an insect-selective toxin derived from a scorpion
(McCutchen et al., 1991), was used for comparative purposes in bioassays.
Binding Assay
JHE-SG was produced in insect cell culture in the absence of FCS, and purified using DEAE anion exchange chromatography as described previously
(Ichinose et al., 1992a). Binding assays were carried out using equilibrium
180
Bonning et al.
dialysis with racemic 3H JH I11 (2.5 x
M final concentration) in PEGcoated glass vials as described elsewhere (Park et al., 1993) with the modification that OTFP was not used. Dialysis tubing was prepared as described
previously (Klotz, 1990). Purified recombinant juvenile hormone binding
protein (rJHBP;Touhara et al., 1993) and P-galactosidase were used as positive and negative controls, respectively. Samples of JHE-SG and P-galactosidase were added to dialysis tubing in excess (10 to 20 pg) compared
to rJHBP (400 ng). Dialysis bags were incubated at 25°C for 16 h with
rotation. The amount of JH bound to each sample was determined by subtracting the concentration of hormone in the external reservoir from that
inside the dialysis bag.
Immunohistochemistry and Western Blot Analysis
The JHE-SG purified by DEAE anion exchange was injected into fourth
stadium larvae of Manduca sexfa (Ichinose et al., 1992b).Control larvae were
injected with BSA. Ten micrograms of sample was injected in each case. One
hour post injection, the pericardial cells were dissected out (Ichinose et al.,
1992b) and placed directly into 0.1% paraformaldehyde in 0.1M phosphate
buffer, pH 7.2, for immunohistochemical analysis. Sections were prepared and
immunogold labeled as described previously (Booth et al., 1992). For Western blot analysis, pericardial cells were placed directly into cracking buffer
following dissection, boiled for 4 min, and run on a 10%SDS polyacrylamide
gel. Proteins were transferred to a nitrocellulose membrane, and detected using the ECL detection kit according to the manufacturer’s instructions (Amersham International plc, Buckinghamshire, U.K.).
Bioassay
Larvae of Trichoplusia ni were maintained as described previously (Bonning
et al., 1992). Larvae of Heliothis virescens were maintained at 26°C on a semisynthetic diet (Hunter et al., 1984; BioServ, Frenchtown, NJ). For determination of lethal times of the recombinant baculoviruses, neonate larvae were
infected at 2,000 pibs/pl using the droplet feeding technique (Hughes et al.,
1986). Mortality was scored every 4,6, or 8 h according to the mortality rate.
The response of each larva to gentle stimulation at the head end using a pipette tip was noted. Larvae which did not respond were scored as dead. Pigmentation of the cuticle, the bloated or contracted nature of the thorax
and abdomen, and partial paralysis were also noted as symptoms. Thirty
larvae were infected per bioassay for each virus, and bioassays were replicated three times. LTs0values were determined using a probit analysis
program which assumes normal distribution and independence of observations (Russell et al., 1977).
Bioassays were also carried out to determine lethal times of recombinant baculoviruses in larvae of H . virescens in conjunction with topical
application of juvenoids, anti-JH agents, or inhibitors of JHE. The doses
used for juvenoids, anti-JH agents, and JHE inhibitors were 30 ng, 5 pg,
and 3 pg, respectively (Bonning and Hammock, unpublished report).
Chemical reagents were suspended in ethanol or acetone and 1 p1 was
applied topically to the dorsal surface of each larva between 4 and 8 h
Baculovirus Expressing Modified JHE
181
post infection. Control larvae were treated with the chemical agent or solvent alone, without virus infection.
For determination of the lethal doses of recombinant viruses, neonate larvae of H. virescens were placed individually into the wells of 24 well plates
(Falcon, Franklin Lake, NJ) with diet for 5 days at 26°C. Larvae that had
reached the third stadium by this time were infected with virus on a diet
plug as described previously (Bonning et al., 1992).Larvae were infected with
one of five doses of virus (3,000, 1,000, 333, 111, 37 pibs) applied previously
to the diet plug in a volume of 1 p1 of distilled water. Only larvae that had
completely consumed the diet plug after 24 h were transferred to cups of
diet and maintained at 26°C. Mortality was scored 10 days later. Thirty larvae were infected at each dose per experiment and bioassays were repeated
three times. Lethal doses were determined using a probit analysis program
(Russell et al., 1977). There is no significant difference in the lethal dose between early and late third stadium larvae of H. virescens (Hoover, unpublished results). Lethal ratios for time (LRT) and dose (LRD) were calculated
at the 50% level from LTs0and LD50 data (Bonning and Hammock, 1993). For
example,
LRTs0=
LTs0of test virus
LT50 of wild type virus.
In Vivo Expression of Modified JHE
In order to assess the expression levels of modified JHEs in vivo, hemolymph
samples from larvae infected with wild-type AcNPV, AcJHE-SG, AcJHE-HK,
AcJHE, AcJHE-RH, and AcJHE-DN (n = 3 or 4 per virus) were run on SDS
polyacrylamide gels and analyzed by Western blot as described above. Larvae of H . virescens were reared to the third stadium and infected as described
above for lethal dose bioassays. Hemolymph samples were taken from a small
incision made in the proleg 3 days post infection for SDS PAGE. Samples
taken from larvae infected with wild-type virus, AcJHE, AcJHE-HK, or AcJHESG (8 to 10 larvae per virus treatment) were also assayed for catalytic activity
using radiolabeled JH (Hammock and Roe, 1985).
Feeding-Damage Bioassay
Newly hatched neonate larvae of H . virescens were dosed at 2,000 pibs/pl
by droplet feeding (Hughes et al., 1986). Thirty larvae were infected with
each test virus. Larvae were transferred individually to inverted 90 mm Petri
dishes containing dampened circles of filter paper and iceburg lettuce (variety Salinas). Lettuce was grown in the greenhouse in the absence of pesticides. Petri dishes were sealed using parafilm and maintained at 26°C. Pieces
of iceburg lettuce (which were not limiting), were changed every 48 h and
the area of each piece scanned before and after feeding using a C1-202 area
meter (CID, Inc., Vancouver, WA). Data for feeding-damage were analyzed
by one-way ANOVA followed by the Scheffe means separation F-test (Steel
and Torrie, 1980) which controls for the experiment-wise type 1 error rate.
Feeding damage assays were also carried out using cotton leaves (variety
182
Bonning et al.
Acala SJ-2). However, because the area of cotton leaves eaten by uninfected
or infected larvae is only about 20% that of lettuce, feeding assays on cotton
were done using third stadium larvae for accurate assessment of differences
in feeding damage between treatments. Larvae were reared to third stadium
(as described above) and starved for 24 h prior to infection with 3,000 pibs
per diet plug. After 16 h, larvae that had completely consumed the diet plug
were transferred to prescanned leaves and the protocol continued as described
above for lettuce.
RESULTS
Binding Assays
Binding assays were carried out using equilibrium dialysis with radiolabeled JH I11 to determine whether the protein JHE-SG will bind JH. No binding of JH to JHE-SG or P-galactosidase was detected despite the presence of
these proteins in excess. A significant amount of JH (18 pMol) was bound to
the rJHBP under the conditions used. This is within the range expected for
racemic JH I11 (Park et al., 1993).
Immunohistochemistry and Western Blot Analysis
Immunohistochemicalanalysis confirmed that when JHE-SG is injected into
larvae of M . sexta, it is removed from the hemolymph by the pericardial cells.
Immunogold labeling revealed concentration of JHE-SG in the lysosome-like
granules within the pericardial cells as seen for wild-type JHE (Booth et al.,
1992).Labeling was above the background levels seen for sections prepared
from larvae injected with BSA. Western blot analysis of pericardial cells taken
from larvae injected with JHE-SG confirmed this result. No JHE was detected
in pericardial cells taken from larvae injected with BSA when analyzed by
Western blot.
Bioassays
Bioassay data for the recombinant baculoviruses expressing JHEs that had
been modified for analysis of the catalytic mechanism (Ward et al., 1992) are
shown in Table 1. No significant differences were seen in time to death of
neonate larvae of H. vivescens except for AcJHE-SG which expresses JHE with
the Ser at the catalytic site mutated to Gly ( P < 0.05). A similar reduction in
lethal time was seen on bioassay of AcJHE-SG in T. ni relative to the wildtype virus (Table 1).
Larvae infected with the wild-type virus were pale and swollen in appearance and lysed rapidly following death of the larva. Larvae infected with the
JHE viruses AcJHE, AcJHE-HK, AcJHE-DN, and AcJHE-RH were pale in appearance at death similar to the wild-type virus-infected larvae (Fig. la), but
lysis was delayed by 24 to 48 h compared to wild type virus-infected larvae.
Of the larvae infected with AcJHE-SG, 27% (n = 159) died at the molt with
two major morphological symptoms. In one case, the larvae showed extensive cuticular blackening and died after head capsule slippage because the
old cuticle failed to split (Fig. lb). Dissection revealed that the larva was
trapped within the old cuticle. In the other case, the old cuticle was not com-
Baculovirus Expressing Modified JHE
183
TABLE 1. Lethal Times of Recombinant Baculoviruses Expressing Modified JHEs in
Trichoplusia ni and Heliothis virescens
Virus
AcNPV C6
AcJHE
AcJHE-SG'
ACJHE-HK~
AcJHE-RH'
ACJHE-DN~
T. ni
LTs0(95%CL)
107 (105-108)
107 (103-111)
90 (87-93)*
-
-
H.virescens
LRT5R
LT5, (95%CLIb
1 .o
1.o
0.84
-
114 (111-117)
118 (114-122)
81 (77-85)*
113 (110-116)
103 (97-108)
94 (85-100)
LRTm
1.0
1.o
0.71
0.97
1.13
1.03
"LRTj0= LTj, recombinant virus
LT5, wild type virus (Bonning and Hammock, 1993).
bData taken from different bioassays.
'Baculovirus expressing JHE with Serzolmutated to Gly.
dBaculovirusexpressing JHE with
mutated to Lys.
'Baculovirus expressing JHE with Arg,, mutated to His.
'Baculovirus expressing JHE with Asp,, mutated to Asn.
*Significantlydifferent from AcNPV C6 ( P < 0.05).
pletely shed (Fig. lc). The remaining larvae infected with AcJHE-SG (73%)
were pale and swollen in appearance similar to larvae infected with AcJHE
(Fig. la). Some of these larvae exhibited contractive paralysis with only partial or no response to stimulation.
Lethal doses for the recombinant baculoviruses in H . virescens were not
significantly different from the wild-type virus ( P > 0.05; Table 2). The high
variance is typical for these bioassays (Bonning et al., 1995).
In Vivo Expression of Modified JHEs
Samples of hemolymph taken from larvae infected with the recombinant
baculoviruses expressing the modified JHEs were analyzed by SDS PAGE
and Western blotting, and assayed for JHE activity. Western blots confirmed
that in vivo expression of the modified forms of JHE was similar to the in
vivo expression of the wild-type JHE. Catalytic activity detected using radiolabeled JH I11 to assay hemolymph taken from larvae infected with wild-type
AcNPV, AcJHE-SG, or AcJHE-HK was not above background levels (5 to 10
nMol JH hydrolyzed/min/ml hemolymph). Activity detected in hemolymph
from larvae infected with AcJHE was between 100 and 150 nMol JH hydrolyzed /min/ml hemolymph.
Feeding-Damage Bioassay
Feeding-damage caused by uninfected neonate larvae (control) and larvae
infected with wild-type AcNPV, AcJHE-SG, or AcAaIT to iceburg lettuce was
compared (Table 3A). Damage caused by the wild-type virus was significantly
less (by 50%) than that caused by uninfected larvae (F = 5.16; P < 0.05; Table
3B). Damage caused by larvae infected with AcJHE-SG was significantly less
(by 66%) than that caused by wild type virus-infected larvae (F = 2.70; P <
0.05), and not significantly different from damage caused by larvae infected
with AcAaIT (F = 0.029; P > 0.05; Table 3B). Damage caused by larvae in-
184
Bonning et al.
Fig. 1 . Symptoms of larvae of H. virescens infected with recombinant baculoviruses expressing
JHEs. a: Larva infected with AcJHE showing characteristic pale coloration. Larvae infected with
the corresponding wild-type virus are also characteristically pale in appearance and lyse rapidly. b: Larva infected with AcJHE-SG showing cuticular blackening. This larva died following
head capsule slippage when the old cuticle failed to split. c: Larva infected with AcJHE-SG
which died after an unsuccessful molt with incomplete shedding of the old cuticle.
TABLE 2. Lethal Doses of Recombinant Baculoviruses Expressing Modified JHEs in
Heliothis virescens
Virus
AcNPV C6
AcJHE
AcJHE-SG
AcJHE-HK
LD50 (95% CL)
106 (73-156)
53 (25-82)
80 (16-170)
47 (22-73)
Baculovirus Expressing Modified JHE
185
TABLE 3A. Feeding Damage to Iceburg Lettuce, and to Cotton Leaves Caused by Larvae of
Heliothis virescens When Infected With Recombinant Baculoviruses Compared to WildType Virus and Uninfected Controls
Mean area consumed" (cm')
Virus
Lettuceb
SD
Cotton'
SD
2.88
1.45
0.49
0.39
1.61
1.50
0.63
0.57
6.92
4.52
2.07
2.92
4.96
2.50
0.90
Control
AcNPV C6
AcJHE-SG
AcAaITd
0.88
an = 30 larvae per treatment.
bFeeding damage caused by neonate larvae.
'Feeding damage caused by third instar larvae.
dBaculovirus expressing an insect-selective scorpion toxin (McCutchen et al., 1991).
TABLE 3B. Statistical Analysis of Feeding Damage Datat
F value
Comparison
Lettuce
Cotton
AcNPV C6 vs. control
AcAaIT vs. control
AcJHE-SG vs. control
AcNPV C6 vs. AcAaIT
AcNPV C6 VS. AcJHE-SG
AcTHE-SG vs. AcAaIT
5.16'
15.78'
14.54'
3.28,
2.70*
0.02
0.04
0.11
0.17
0.02
0.04
0.05
'Groups compared by one-way ANOVA followed by the Scheffe means separation F-test.
*Significantlydifferent ( P < 0.05).
fected with AcJHE was not significantly different to damage caused by the
wild-type virus (data not shown). Similar results were seen for feeding bioassays carried out on cotton with third stadium larvae (Table 3A; Fig. 2). On
cotton, damage caused by wild-type AcNPV-infected third stadium larvae
was 35% less than that caused by uninfected larvae, and damage caused
by larvae infected with AcJHE-SG was 54% less than that caused by
AcNPV-infected larvae (Table 3A). However, the greater variability in feeding-damage caused by third stadium larvae (which is not seen for neonate larvae) was such that none of the comparisons between treatments
on cotton showed significant differences when compared by one-way
ANOVA and the Scheffe means separation F test (Table 38). This was also
seen for feeding-damage bioassays with third stadium larvae on iceburg
lettuce (data not shown).
Tables 4 to 6 show data for bioassays of wild-type and recombinant viruses
in combination with various chemical agents designed to disrupt JH regulation. Table 4 shows data for the wild-type AcNPV. This table shows that two
structurally distinct juvenoids and two separate chemical classes of JHE inhibitors tend to decrease the LTs0of the wild-type virus on infection and treatment of neonate larvae, while anti-JH agents tend to increase the lethal time.
This may be related to the effects of JH and juvenoids on the fat body which
is the primary site of virus replication (Bonning and Hammock, unpublished
186
Bonning et a!.
Fig. 2. Feeding damage on cotton caused by an uninfected third stadium larva of H. virescens
(control) or larvae infected with wild-type AcNPV (WT AcMNPV), or AcJHE-SG (S201G AcMNPV).
An undamaged leaf (untreated) i s shown for comparison. Damage caused by w i l d type virusinfected larvae is 35% less than that caused by uninfected larvae. Damage caused by AcJHE%-infected larvae i s 54% less than that caused by wild-type virus-infected larvae (Table 3).
report). Table 5 shows bioassay data for AcJHE. The juvenoids and JHE inhibitors increase the LT50where significant differences are seen between treated
and untreated larvae infected with AcJHE ( P < 0.05). Greater variation was
seen in data for the anti-JH agents, with only one out of six data sets being
significantly different. In this case the lethal time of AcJHE was increased by
FMev. Table 6 shows data for AcJHE-SG. In contrast to the wild-type virus,
the juvenoids and JHE inhibitors increase the lethal time of AcJHE-SG as
they do with AcJHE. The anti-JH agents FMev and ETB increase the lethal
time of AcJHE-SG fairly consistently, as they do for the wild-type virus. These
data are not consistent with the hypothesis that AcJHE-SG acts by reducing
the JH titer. No mortality was observed for uninfected larvae treated with
the chemical agents or solvents alone.
Baculovirus Expressing Modified JHE
187
TABLE 4. Effect of Various Chemical Agents on the Lethal Time of Wild-Type AcNPV on
Infection of Neonate Larvae of H. zlirescelzs*
LTSo (95% CL)
Treatment
JH analogs
Epofenonane
Fenoxycarb
JHE inhibitors
OTFP
EPPAT
Anti-JH agents
FMev
ETB
Control
Treated
162 (157-168)
124 (120-129)
108 (103-111)
163 (156-1 70)
162 (157-168)
124 (120-129)
108 (103-111)
163 (156-170)
146 (139-155)”
108 (103-113)“
97 (95-100)”
148 (137-162)
167 (158-178)
117 (112-122)
106 (101-109)
142 (133-151)”
162 (157-168)
131 (126-336)
131 (125-135)
100 (94-105)
131 (126-136)
164 (161-168)
121 (118-125)”
104 (98-109)”
101 (98-105)
115 (105-125)’
133 (122-143)
146 (139-153)
115 (111-119)
137 (132-141)
145 (139-150)
144 (139-149)
144 (139-149)
131 (125-138)
166 (158-175)”
118 (112-123)
158 (150-166)”
149 (144-155)
161 (155-167)”
154 (150-159)”
*No mortality was seen in the uninfected control larvae treated with solvent or the chemical
agent alone.
”Significantlydifferent at 95% CL.
DISCUSSION
We have developed a fast-acting recombinant baculovirus which expresses
a modified form of JHE which is inactive with respect to its function of JH
catalysis. The most obvious hypothesis for the action of AcJHE-SG is that it
reduces the effective JH titer at the tissue level synonymous with the function of the wild-type JHE. It follows from this hypothesis that both the injected recombinant protein (JHE-SG)and the recombinant baculovirus would
act as biochemical anti-JH agents. Several lines of evidence refute this hypothesis. First the catalytic activity of JHE-SG is dramatically reduced in vitro
(Ward et al., 1992) making it very unlikely that the ex ressed enzyme in infected insects is converting JH to JH acid. Assays with H-JH showed that the
catalytic activity of THE-SG was reduced > lo6 fold over that of recombinant
wild-type JHE (Ward et al., 1992). A second hypothesis is that expression of
high levels of JHE-SG spare the natural JHE from degradation. In this case,
the JH titer would be reduced by the natural THE. This hypothesis is not
supported by the fact that the stage of insects treated is not anticipated to
have high levels of endogenous JHE (Hammock, 1985). In addition, the
hemolymph from insects infected with AcJHE-SG did not show significant
catalytic activity on JH, in contrast to hemolymph from larvae infected with
9
188
Bonning et al.
TABLE 5. Effect of Various Agents on the LTs0of AcJHE on Infection of Neonate Larvae of
H . virescens
LT50 (95% CL)
Treatment
JH analogs
Epofenonane
Fenoxycarb
JHE inhibitors
OTFP
EPPAT
Anti-JH agents
FMev
ETB
LRT5t
Control
Treated
1.02
1.02
1.01
1.02
0.98
1.02
111 (108-114)
104 (98-108)
102 (93-108)
97 (90-106)
145 (137-155)
104 (98-108)
109 (106-113)
118 (112-125)b
126 (119-133)b
111 (108-114)b
147 (140-155)
125 (118-131)b
1.02
1.01
1.02
0.97
1.04
111 (108-114)
102 (93-108)
104 (98-108)
131 (127-135)
107 (104-110)
108 (104-113)
120 (114-125)b
119 (lll-126)b
153 (145-162)b
111 (104-118)
0.95
0.97
1.02
1.04
1.04
1.02
127 (121-133)
131 (127-135)
104 (98-108)
107 (104-110)
107 (104-110)
105 (101-109)
127 (120-133)
122 (117-127)
116 (109-123)b
110 (106-114)
115 (106-124)
107 (103-111)
aLRT50= LTs0recombinant virus
LTs0wild type virus.
bSignificantlydifferent at 95% CL.
AcJHE which expresses the catalytically active enzyme. The pharmacological
data discussed below also argue against this hypothesis. A third hypothesis
is that the THE-SG has lost its ability to catalyze JH but still binds the hormone tightly enough to sequester it. For this hypothesis to be correct, the
protein must have a high affinity for JH and be at a high enough concentration in vivo to effectively prevent JH from binding to its receptor. We have a
good model for comparison in the hemolymph JH binding protein (JHBP).
This protein is at relatively high concentration in the hemolymph of both
Manduca sexta (Goodman and Gilbert, 1978; Hidayat and Goodman, 1994)
and T. ni (Wing et al., 1981) yet is compatible with normal insect metamorphosis. Thus, for JHE-SG to act by this last mechanism it would have to be at
significantly higher concentration and/or bind JH with much higher affinity
than the natural JHBP. As a control, the JHBP from M.sexta (Touhara et al.,
1993) was expressed in the baculovirus system. The resulting virus failed to
show any significant effects on the speed of kill or symptoms of infected
larvae although production of this protein by the baculovirus system was
quite high (50 mg 1-' in Sf21 cell culture; Bonning and Touhara, unpublished
results). Under conditions where the recombinant JHBPbound JH 111, no binding to THE-SG was detected. We interpret these data to suggest first that the
K, of the wild-type enzyme (Abdel-Aal et al., 1988) is largely the result of
catalytic events leading to the formation of the acyl enzyme or that the
Baculovirus Expressing Modified JHE
189
TABLE 6. Effect of Various Agents on the LTs0of AcJHE-SG on Infection of Neonate
Larvae of H.virescens
LTso (95% CL)
Treatment
JH analogs
Epofenonane
Fenoxycarb
JHE inhibitors
OTFP
EPPAT
Anti-JH agents
FMev
ETB
LRTma
0.75
0.74
0.84
0.75
0.74
0.84
0.75
0.73
0.73
0.73
0.78
0.83
0.82
0.88
0.86
0.86
Control
93
122
102
93
122
102
Treated
(88-98)
(116-129)
(98-106)
(88-98)
(116-129)
(98-106)
105
147
104
92
140
109
(101-109)b
(141-152)b
(100-109)
(87-97)
(134-147)b
(102-116)
93 (88-98)
94 (84-102)
94 (82-102)
94 (84-102)
102
105
101
100
(98-106)b
(98-110)
(94-106)
(93-107)
130
143
105
143
100
91
(122-137)b
(136-149)b
(100-109)
(137-148)b
(95-106)b
(87-95)
105
126
103
128
88
88
(98-111)
(121-132)
(98-107)
(122-134)
(84-92)
(84-92)
aLRT50= LTsorecombinant virus
LTs0wild type virus.
bSignificantlydifferent at 95% CL.
protein’s structure is so altered by Gly,,, that it no longer binds JH. The JHESG protein does not appear to be at a higher concentration in the hemolymph
of larvae infected with AcJHE-SG than wild-type JHE in larvae infected with
AcJHE. Western blots of the hemolymph of insects infected with AcJHE, AcJHESG, and other mutants show similar levels of recombinant proteins. The bioassay data discussed below also argue against the JH binding hypothesis.
The best argument against the catalytic mechanism of AcJHE-SG directly
involving JH comes from the biological assays described here. Experiments
based on the use of two structurally distinct juvenoids and two structurally
distinct JHE inhibitors support the hypothesis that the decrease in lethal time
of AcJHE-SG is not the result of an anti-JH mechanism. Anti-JH agents would
be expected to decrease the lethal time of a recombinant with an anti-JH action, rather than increase the lethal time as seen for AcJHE-SG. Chemical antiJH agents would thus be expected to show additive and/or synergistic effects
with a recombinant virus showing anti-JH effects which would lead to more
rapid kill. This has been shown for another modified JHE which has been
stabilized against degradation and expressed in a baculovims for insect control (Bonning et al., unpublished report).
JHE is exported from the baculovirus-infected cell (Bonning et al., 1992;
Hammock et al., 1990) into the hemolymph of the larva. From here it is removed by an active uptake process by the pericardial cells (Booth et al., 1992;
Ichinose et al., 1992a,b). Using JHE-SG purified by DEAE anion exchange,
190
Bonning et al.
we have shown using Western blotting and electron microscopy, that alteration of the catalytic site serine has not affected uptake of the protein by the
pericardial cells.
The following three hypotheses for the mechanism of AcJHE-SG are exclusive of JH. First, expression of JHE-SG by the recombinant virus could be
saturating the pericardial cells resulting in disruption of homeostasis. This
hypothesis is not supported by data for other recombinant baculoviruses
which, when injected into larvae for very high levels of infection, express
wild-type JHE to extremely high levels in the hemolymph without any toxic
effect (for review see Bonning and Hammock, 1994). Secondly, the normal
process of degradation of the enzyme within the pericardial cells could have
been disrupted by alteration of the serine residue at the catalytic site. This
hypothesis is currently under investigation. Finally, proteins which are structurally related to esterases have a number of roles unrelated to catalytic activity. These include both structural and transport functions (Cygler et al.,
1993; Ollis et al., 1992). It is conceivable that by replacing the SerZolof JHE,
we have inadvertently generated a protein which has such properties.
Events at the larval molt appear to be particularly susceptible to the effects
of JHE-SG. The exact mechanism whereby the old cuticle splits at the start of
the molt is unknown. Failure of the cuticle of AcJHE-SG infected larvae to
split may be related to generation of inadequate fluid pressure which facilitates molting. Failure to escape completely from the old cuticle may be related to the contraction paralysis seen in some specimens. Partial paralysis would
hinder the muscular movements associated with escape from the exuvium.
It cannot be assumed that a reduced lethal time of a recombinant baculovirus is proportional to the resultant feeding damage. Larvae in bioassays are
scored as dead only when no response is observed in reaction to stimulation
at the head end. Hence, if larvae are trapped at the molt, or partially paralyzed, the lethal time will underestimate the reduction in feeding damage.
The variation in LRTs0for AcJHE-SG (from 0.73 to 0.88) can be seen in Table 6
and may be related to the exact age of neonate larvae at the time of infection.
This is currently under investigation. Despite this variation, feeding damage
on iceburg lettuce was not significantly different from that caused by AcAaIT
which kills fairly consistently with an LRT50of 0.7 (Bonning and Hammock,
1993; McCutchen et al., 1991; Stewart et al., 1991).Larvae infected with AcJHESG remain on the plant but become sluggish and stop feeding, whereas larvae infected with AcAaIT fall off the plant as a result of rapid paralysis (Cory
et al., 1994). The fact that cadavers of larvae infected with AcJHE-SG remain
on the plant will facilitate dispersal of the virus following liquefaction of the
cadaver, but may not be so appealing from the perspective of the farmer or
consumer.
We have demonstrated the potential of the recombinant baculovirus AcJHESG as an effective insecticidal agent. A significant reduction in feeding damage is seen on infection of larvae with AcJHE-SG which is comparable to that
for AcAaIT, widely seen as the most effective recombinant baculovirus with
reduced lethal time developed to date (McCutchen et al., 1991; Stewart et al,
1991). However, neither recombinant virus reduces feeding damage sufficiently to warrant commercialization. Additives or synergists used in con-
Baculovirus Expressing Modified JHE
191
junction with these recombinant baculoviruses (McCutchen et al., 1994;
Shapiro and Dougherty, 1993) can be expected to facilitate their efficacy as
crop protection agents. An understanding of the mechanism by which JHESSG kills larvae, and the pharmacokinetics of the recombinant protein may
facilitate development of recombinant viruses with a greater reduction in the
lethal time on infection of pest insects.
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