Insect Control: Biological and Synthetic Agents - Index of

10.2.2.2. Gene silencing and RNA interference
AcPR1 is a recombinant AcMNPV that produces
biologically active JHE of H. virescens (Hanzlik
et al., 1989) under the very late baculoviral p10
gene promoter (Roelvink et al., 1992). AcPR2 is a
recombinant AcMNPV in which the same JHE gene
is placed under the p10 promoter in the antisense
direction. Transcription of this gene generates
mRNAs that are antisense to the JHE mRNAs generated
by AcPR1. These antisense JHE mRNAs are
able to reduce the JHE activity produced by AcPR1
when AcPR1 and AcPR2 are coinfected into Spodoptera
frugiperda (Roelvink et al., 1992). By injection
of AcPR2 into fifth instar H. virescens, Hajos
et al. (1999) showed that AcPR2 downregulates
JHE activity in more than 95% of the injected
larvae. This effect was putatively due to direct
RNA–RNA interaction between sense and antisense
JHE RNAs. Although 95% of the infected larvae
showed a reduction in JHE activity, only 25%
of these larvae showed morphogenic alterations
(Hajos et al., 1999). These alterations were similar
to those induced by the exogenous application of
JH (Cymborowski and Zimowska, 1984) or JHE
inhibitors (Hammock et al., 1984) to the larvae.
On the basis of the effectiveness of this baculovirus
mediated gene silencing approach, Hajos et al.
(1999) suggest that the identification of other host
gene targets could greatly reduce lethal times or
feeding damage of a GM baculovirus pesticide.
RNA interference (RNAi) is a sequence specific
mechanism by which a targeted gene is silenced by
the introduction of double-stranded RNA (dsRNA)
that is homologous to the targeted gene (Hutvagner
and Zamore, 2002; Denli and Hannon, 2003).
Mechanistically, it is likely that the antisense RNA
10: Genetically Modified Baculoviruses for Pest Insect Control 337
mediated gene silencing described by Hajos et al.
(1999) involves the RNAi pathway. RNAi has
been demonstrated in a diverse range of organisms
including plants, fungi, arthropods, and mammals.
One biological function of RNAi appears to be as a
defense mechanism against RNA viruses in plants
and other organisms. Recently, RNAi has been
shown to be effective in lepidopteran cell lines
(Means et al., 2003; Valdes et al., 2003) and larvae
such as the giant silk moth, H. cecropia (Bettencourt
et al., 2002) and cabbage looper T. ni (Kramer and
Bentley, 2003). Our laboratory has explored the use
of RNAi to block the activity and effects of JHE in
cultured S. frugiperda cells and larvae of H. virescens.
In the larval experiments, a 0.5 kb dsRNA
fragment that corresponds to the 5 0 -coding sequence
of the JHE gene of H. virescens (Hanzlik et al.,
1989) was generated. Neonate H. virescens that
ingested these dsRNAs within 6 h of hatching
showed a statistically significant (two-sided Mann-
Whitney test with an error type I of 99%) increase in
weight (in comparison to control larvae) by the
second and third days of the fifth larval instar.
Additionally, injection of the same 0.5 kb dsRNAs
into the larvae of H. virescens on the first day of the
fifth instar resulted in a 16 h delay in the median
time to pupation in comparison to water injected or
untreated controls (Figure 2). Treatments were
compared with Wilcoxon (Gehan) statistics on a
99% error type I. These comparisons indicated
that RNAi against the JHE of H. virescens is effective
at prolonging the juvenile state. The authors are
currently in the process of constructing recombinant
AcMNPVs carrying JHE gene derived sequences in
a ‘‘head-to-head’’ manner such that the expressed
RNAs will form a hairpin loop structure. Such a
Figure 2 Percentage of larvae that pupate over time following treatment with double-stranded RNA (dsRNA), double-distilled
water, or untreated. Pupation was scored on the basis of head capsule slippage at 1.5 h or longer intervals.