Insect Control: Biological and Synthetic Agents - Index of

344 10: Genetically Modified Baculoviruses for Pest Insect Control
H. armigera (HaSNPV; Chen et al., 2000; Sun et al.,
2002, 2004). RoMNPV is also known as Anagrapha
falcifera Kirby MNPV (Harrison and Bonning,
1999). Harrison and Bonning (2000b) have generated
recombinant RoMNPVs (Ro6.9AaIT) expressing
AaIT fused to the bombyxin signal sequence
under the late p6.9 promoter of AcMNPV. The
p6.9 promoter, like the very late promoters, requires
the products of viral early genes and the initiation
of viral DNA replication for its activation (Lu
and Milller, 1997). However, it is activated earlier
and drives higher levels of expression than the
p10 or polh promoters in tissue culture (Hill-Perkins
and Possee, 1990; Bonning et al., 1994). In dosemortality
bioassays (droplet feeding), there were
no significant differences between the LC50s of
Ro6.9AaIT and the wild-type RoMNPV in neonate
European corn borer Ostrinia nubilalis (8.1 10 6
and 1.0 10 7 polyhedra per ml, respectively) or
H. zea (9.5 10 4 and 5.4 10 4 polyhedra per ml,
respectively). The LC 50s, however, were significantly
different in neonate H. virescens (1.67 10 5 and
8.4 10 4 polyhedra per ml, respectively). Survivaltime
bioassays (at an LC99 dose) of neonates of O.
nubilalis, H. zea, and H. virescens infected with
Ro6.9AaIT generated ST50s that were reduced by
approximately 34% (120 versus 181 h), 37% (81
versus 128 h), and 19% (72.5 versus 89.5 h), respectively,
in comparison to control larvae infected
with RoMNPV. Later in this chapter, recombinant
HzNPV and HaSNPV constructs in which the AaIT
gene was placed under a very late promoter or other
alternative promoter (e.g., early, chimeric, or constitutive)
and inserted at the egt gene locus of the virus
are discussed.
10.3.1.2. Lqh and Lqq A series of highly potent
insecticidal toxins have been identified and characterized
from the venom of yellow Israeli scorpions,
L. quinquestriatus hebraeus and L. quinquestriatus
quinquestriatus. Both excitatory (e.g., LqqIT1,
LqhIT1, and LqhIT5) and depressant (e.g., LqhIT2
and LqqIT2) insect selective toxins have been
isolated from these scorpions (Zlotkin et al.,
1985, 1993; Kopeyan et al., 1990; Zlotkin, 1991;
Moskowitz et al., 1998). Gershburg et al. (1998)
have generated recombinant AcMNPVs expressing
the excitatory LqhIT1 toxin under the very late p10
(AcLIT1.p10) and early p35 (AcLIT1.p35) gene promoters,
and the depressant LqhIT2 toxin under the
very late polh gene promoter (AcLIT2.pol). These
constructs expressed LqhIT1 and LqhIT2 that were
secreted to the outside of the cell under endogenous
secretion signals. On the basis of time-mortality
bioassays using neonate H. armigera, they found
that the median effective times (ET50s) for paralysis
and/or death of AcLIT1.p10 and AcLIT2.pol were
66 and 59 h. This was an improvement of roughly
24% and 32%, respectively, in comparison to the
wild-type AcMNPV (ET 50 of 87 h). The ET 50 was
also reduced by about 16% (to 73 h) when LqhIT1
was expressed under the early promoter by
AcLIT1.p35, although not as dramatically in comparison
to AcLIT1.p10. Imai et al. (2000) have
generated a recombinant BmNPV (BmLqhIT2)
expressing LqhIT2 fused to a bombyxin signal sequence
under the polh gene promoter. Fourth instar
larvae of B. mori that were injected with BmLqhIT2
(2 10 5 pfu per larva) initially showed LqhIT2 specific
symptoms at 48 h post injection and stopped
feeding at roughly 82 h post injection. This was an
improvement of roughly 35% in the ET50 in comparison
to the wild-type BmNPV.
Harrison and Bonning (2000b) have generated
recombinant AcMNPVs expressing LqhIT2 fused
to a bombyxin signal sequence under the late p6.9
(AcMLF9.LqhIT2) or very late p10 (AcUW21.
LqhIT2) gene promoters. Survival-time bioassays
(at an LC99 dose) of neonate H. virescens infected
with AcMLF9.LqhIT2 or AcUW21.LqhIT2 showed
that the ST50s (63.5 h) of larvae infected with these
viruses were the same (Harrison and Bonning,
2000b). However, log-rank tests of the Kaplan-
Meier survival curves of larvae infected by
these viruses were significantly different (P < 0.05).
In comparison to AcUW21.LqhIT2 infected neonate
H. virescens, more of the AcMLF9.LqhIT2
infected neonates died at earlier times postinfection
(Harrison and Bonning, personal communication).
In comparison to control larvae infected with
wild-type AcMNPV, AcMLF9.AaIT (recombinant
AcMNPV expressing AaIT under the p6.9 gene promoter),
or AcAaIT, neonate H. virescens infected
with AcMLF9.LqhIT2 or AcUW21.LqhIT2 showed
reductions in median survival times of approximately
34% (63.5 versus 96.0 h), 10% (63.5 versus
71.0 h), and 10% (63.5 versus 70.5 h), respectively.
Harrison and Bonning (2000b) have also generated
recombinant RoMNPVs expressing LqhIT2 under
the p6.9 (Ro6.9LqhIT2) and p10 (Ro10LqhIT2)
promoters of AcMNPV. Ro10LqhIT2 produced
visibly fewer polyhedra that potentially occluded
fewer virions in Sf-9 cell cultures and thus were
not used in bioassays. Survival-time bioassays
(at an LC99 dose) in neonate larvae of the European
corn borer O. nubilalis Hübner, H. zea, and
H. virescens infected with Ro6.9LqhIT2 generated
ST 50s that were approximately 41% (107 versus
181 h), 42% (74.5 versus 128 h), and 27% (65.5
versus 89.5 h) faster, respectively, than those