Insect Control: Biological and Synthetic Agents - Index of

generated in control neonates infected with wildtype
RoMNPV. The ST50s of Ro6.9LqhIT2 infected
neonate H. zea and H. virescens were also significantly
lower than control neonates infected with a
recombinant RoMNPV expressing AaIT under the
p6.9 promoter.
Chejanovsky et al. (1995) have generated a
recombinant AcMNPV (AcLa22) that expresses the
L. quinquestriatus hebraeus derived alpha toxin,
LqhaIT (Eitan et al., 1990). The LT50 of AcLa22
was roughly 35% faster (78 versus 120 h) than that
of the wild-type AcMNPV in larvae of H. armigera.
Since the LqhaIT toxin binds at a different site on
the insect sodium channel from that of the excitatory
toxins (Zlotkin et al., 1978; Cestele and Catterall,
2000), Chejanovsky et al. (1995) suggested that a
baculovirus expressing both alpha and excitatory
toxins may yield a synergistic interaction between
the toxins. However, they cautioned that LqhaIT
toxin lacks absolute selectivity for insects (Eitan
et al., 1990); thus, a recombinant baculovirus
expressing LqhaIT is not appropriate as a biological
pesticide. The LqhaIT toxin, however, should be an
effective tool to study the targeting of different types
of toxins on the voltage gated sodium channel. The
effectiveness of the use of multiple synergistic toxins
is discussed below. Other examples of the expression
of insect selective toxins from L. quinquestriatus
hebraeus using alternative promoters, signal
sequences for secretion, viral vectors, and/or insertion
of the toxin gene at the egt gene locus are given
later in this chapter.
10.3.2. Mite Toxins
The insect-predatory straw itch mite Pyemotes tritici
encodes an insect paralytic neurotoxin TxP-I
that induces rapid, muscle contracting paralysis in
larvae of the greater wax moth G. mellonella
(Tomalski et al., 1988, 1989). TxP-I is encoded by
the tox34 gene as a precursor protein of 291 amino
acid residues. The mature protein is secreted from
the insect cell following cleavage of a 39 amino acid
long signal sequence for secretion (Tomalski and
Miller, 1991). The mode of action of TxP-I is unknown,
however, it is highly toxic (even at a dose of
500 mgkg 1 ) to lepidopteran larvae but not toxic
to mice at a dose of 50 mg kg 1 . A recombinant,
occlusion-negative AcMNPV (vEV-Tox34) expressing
the tox34 gene under a modified polyhedrin
promoter (PLSXIV; Ooi et al., 1989) was shown
to paralyze or kill fifth instar larvae of T. ni by
2 days post injection with 400 000 pfu of BV. In
contrast, control larvae injected with BV of wildtype
AcMNPV never showed symptoms of paralysis
(Tomalski and Miller, 1991). Tomalski and Miller
10: Genetically Modified Baculoviruses for Pest Insect Control 345
(1991, 1992) have constructed two other occlusionnegative
AcMNPVs that express the tox34 gene
under early (vETL-Tox34) or hybrid late/very late
(vCappolh-Tox34) gene promoters as well as an
occlusion-positive AcMNPV (vSp-Tox34) expressing
tox34 under a different hybrid late/very late
promoter. The activities of these constructs will
be discussed later in this chapter. Lu et al. (1996)
have constructed an occlusion-positive AcMNPV
(vp6.9tox34) that utilizes the late p6.9 promoter
to drive expression of tox34. Use of this late promoter
resulted in earlier, by at least 24 h, and higher
level of TxP-I expression in comparison to TxP-I
expression under a very late promoter. As discussed
above, the p6.9 gene promoter is not an
early promoter, but is activated earlier and can
drive higher levels of expression than very late promoters
in tissue culture (Hill-Perkins and Possee,
1990; Bonning et al., 1994). In time-mortality
bioassays (at an LC95 dose), the median time to
effect (ET 50) of vp6.9tox34 in neonate larvae of
S. frugiperda and T. ni was reduced by approximately
56% (44.7 versus 101.3 h) and 58% (41.7
versus 99.0 h), respectively, in comparison to wildtype
AcMNPV. In neonate S. frugiperda and T. ni,
the earlier and higher level of TxP-I expression
under the late promoter resulted in a 20–30% faster
induction of effective paralysis or death in comparison
to TxP-I expression under the very late gene
promoter.
Burden et al. (2000) have constructed a slightly
different TxP-I encoding gene (tox34.4) by RT-PCR
of mRNAs purified from total RNA extracted from
P. tritici using primers designed to amplify the tox34
open reading of Tomalski and Miller (1991). A
recombinant AcMNPV (AcTOX34.4) expressing
TxP-I under the p10 promoter has been generated.
The LD50s of AcTOX34.4 were not significantly
different than those of the wild-type AcMNPV in
dose-mortality bioassays of second (9.3 polyhedra
per larva) and fourth (13.1 polyhedra per larva)
instar larvae of T. ni. In time-mortality bioassays,
second and fourth instar larvae of T. ni infected with
AcTOX34.4 showed a 50–60% reduction (depending
on virus dose and instar) in the mean time to
death in comparison to control larvae infected with
wild-type AcMNPV. There was also a dramatic
reduction in the yield of progeny virus (number of
polyhedra per mg of cadaver) at the time of death.
Second and fourth instar larvae of T. ni that were
infected with AcTOX34.4 produced roughly 85%
and 95% lower yields of polyhedra per unit
weight, respectively, in comparison to control larvae
infected with AcMNPV. On the basis of pathogen–
host model systems that describe how insect viruses