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