Insect Control: Biological and Synthetic Agents - Index of

342 10: Genetically Modified Baculoviruses for Pest Insect Control
main groups, are further classified into mammal or
insect selective toxins, with the insect selective toxins
being further classified on the basis of their mode
of action into three subgroups: alpha insect toxins,
excitatory toxins, and depressant toxins (Zlotkin
et al., 1995; Gordon et al., 1998; Inceoglu et al.,
2001b). Each of these subgroups target different
molecular sites and produce distinct effects upon
the voltage gated sodium channel of the insect
(Cestele and Catterall, 2000). In addition, when
injected into larvae of the blowfly S. falculata,
toxins in each of these subgroups display unique
symptoms. Alpha insect toxins cause contractive
paralysis that is delayed and sustained (Eitan et al.,
1990). Excitatory toxins, such as AaIT from the
North African scorpion Androctonus australis,
cause paralysis that is immediate and contractive
(Zlotkin et al., 1971, 1985; Zlotkin, 1991). In contrast,
depressant toxins, such as LqhIT2 from the
yellow Israeli scorpion Leiurus quinquestriatus
hebraeus, cause transient (until 5 min post injection)
contractive paralysis, followed by sustained, flaccid
paralysis (Zlotkin et al., 1985; Zlotkin, 1991).
Carbonell et al. (1988) were the first to attempt to
express biologically active scorpion toxin, insectotoxin-1
of Buthus eupeus (BeIt), under control of
the very late polyhedrin promoter using recombinant
AcMNPV constructs (vBeIt-1, vBeIt-2, and vBeIt-3).
The vBeIt-1 construct carried the 112 nucleotidelong
BeIt gene 6 nucleotides downstream of
the last nucleotide of the polyhedrin leader. The
vBeIt-2 construct carried the BeIt gene fused to a 21
amino acid-long signal sequence for secretion of
human b-interferon (Ohno and Taniguchi, 1981) 18
nucleotides downstream of the polyhedrin leader.
The vBeIt-3 construct expressed BeIt as a fusion
with the N-terminal 58 codons of the polyhedrin
gene. All three constructs produced similar, high
levels of BeIt specific transcripts from the polyhedrin
promoter. The vBeIt-1 and vBeIt-2 constructs produced
exceptionally low levels of the 4 kDa BeIt
peptide, whereas the vBeIt-3 construct produced
significant amounts of a 13–14 kDa polyhedrin-BeIt
fusion peptide. Toxin specific biological activity,
however, was not observed by bioassay in larvae
of T. ni, Galleria mellonella, and Sarcophaga.
Carbonell et al. (1988) speculated that this was the
result of: (1) toxin instability; (2) a low sensitivity
threshold of the toxin bioassay; and/or (3) inability
of the polyhedrin-BeIt fusion to properly fold.
10.3.1.1. AaIT The insect-selective neurotoxin
AaIT (Androctonus australis insect toxin 1), found
in the venom of the scorpion A. australis, was the
first scorpion toxin to be expressed by recombinant
baculoviruses that showed biological activity
(Maeda et al., 1991; McCutchen et al., 1991;
Stewart et al., 1991). AaIT is composed of a single
polypeptide chain of 70 amino acid residues crosslinked
by four disulfide bonds and is highly specific
for the voltage gated sodium channel of insects
(Zlotkin et al., 2000). The toxin induces a neurological
response similar to that evoked by the pyrethroid
insecticides, but apparently acts at a
different site within the sodium channel (see discussion
below regarding toxin interactions with chemical
pesticides). Maeda et al. (1991) constructed a
recombinant BmNPV carrying a synthetic AaIT
gene (Darbon et al., 1982) that was linked to a
signal sequence for secretion of bombyxin and
driven by the very late polyhedrin (polh) gene promoter.
The recombinant virus, BmAaIT, expressed
biologically active AaIT that was secreted into the
hemolymph of BmAaIT infected silkworm larvae.
The BmAaIT infected larvae (second to fifth instar
B. mori) displayed symptoms that were consistent
with sodium channel binding by AaIT including
body tremors, dorsal arching, feeding cessation,
and paralysis beginning at 40 h p.i. Death occurred
by 60 h p.i. This corresponded to an improvement
in speed of kill of approximately 40% in comparison
to control larvae infected with the wild-type
BmNPV.
In related experiments, McCutchen et al. (1991)
and Stewart et al. (1991) independently generated
recombinant AcMNPVs expressing AaIT under the
very late baculoviral p10 promoter. The Stewart
et al. construct, AcST-3, expressed AaIT as a fusion
protein with the signal sequence of the baculoviral
GP67 protein. The McCutchen et al. construct,
AcAaIT, expressed AaIT as a fusion protein with
the bombyxin signal sequence. Bioassays using orally
inoculated, second instar larvae of T. ni indicated
that the median lethal dose (LD50) of AcST-3 was
reduced by about 30% (44 versus 31 polyhedra per
larva) in comparison to the wild-type AcMNPV. The
median survival time (ST50) ofAcST-3infectedlarvae
(infected with approximately 17.4 polyhedra per
larva) was reduced by about 25% (85.8 versus
113.1 h) in comparison to control larvae infected with
AcMNPV. Third instar T. ni that are infected with a dose
of AcST-3 that resulted in 100% mortality showed
a 50% reduction in feeding damage in comparison
to AcMNPV infected control larvae. The McCutchen
et al. construct, AcAaIT, showed similar results
by bioassay using second instar larvae of H. virescens.
The LD50 of AcAaIT was reduced by about
39% (13.3 versus 21.9 polyhedra per larva)
in comparison to AcMNPV. The ST 50 of AcAaIT
infected larvae (infected with 250 polyhedra per