Insect Control: Biological and Synthetic Agents - Index of

eing associated with an accumulation of neurosecretory
material in the corpora cardiaca and
brain (Subrahmanyam et al., 1989). In R. prolixus
reduction in oocyte growth and egg production
are caused also by reduced ecdysteroid levels in
hemolymph and ovaries (Feder et al., 1988; Garcia
et al., 1991). In the cotton stainer bug Dysdercus
koenigii and the peach potato aphid Myzus persicae
azadirachtin applied topically or in the diet inhibits
and delays embryogenesis, resulting either in many
of the embryos remaining at a late stage of development
or in the viviparous aphid being born dead
with undeveloped appendages (Koul, 1984; Nisbet
et al., 1994). It is thought that JH control of embryogenesis
and the rate of development of embryos
in aphids are important here.
In adult female earwigs, Labidura riparia, azadirachtin
has been shown to have marked cytological
effects on ovaries resulting in severely reduced
ovarian development. Due to cyclical reproductive
changes in L. riparia throughout adult life, relating
to the production of eggs (vitellogenesis) and a
nonvitellogenic period when the female cares for
her eggs, there was the possibility of investigating
the specific role of azadirachtin in vitellogenesis
control. In this insect JH controls both vitellogenin
production in the fat body and stimulates vitellogenin
uptake by the ovaries. In addition, ovarian
ecdysteroids play a role in vitellogenesis and, at
the peak of production, oviposition. They also
provide a feedback loop to the brain pars lateralis
neurosecretory cells causing release of allatostatins,
which block JH synthesis and release enabling
successive follicle degenerations during the nonvitellogenic
period. Azadirachtin treatment blocks
vitellogenesis and corpus allatum activity as seen
by ultrastructural studies and concomitantly
increases allatostatin build-up in the pars lateralis
as measured by antibodies to allatostatin 3 (Blattella
germanica BLAST-3) (Sayah et al., 1996, 1998).
This phenomenon together with the fact that JH
treatment rescues the effect of azadirachtin on
vitellogenesis suggest that azadirachtin affects those
peptides controlling corpus allatum activity in a
similar manner to the azadirachtin effects on PTTH
and hence ecdysone production during the molt.
Two major conclusions can be drawn regarding
the mode of action of azadirachtin on neuroendocrine
activity in insects during the molt and
reproductive development. First, in both molting
and sterility, the processes of synthesis, transport,
and release of morphogenetic peptide hormones in
the brain are at the center of the mode of action
of azadirachtin. Second, in the molt (but not
yet proved during reproductive development) the
5: Azadirachtin, a Natural Product in Insect Control 195
production of important enzymes for controlling
hemolymph levels of active hormones (20-hydroxymonooxygenase
levels that convert ecdysone to
20E) are inhibited by azadirachtin.
5.7. Studies on the Mode of
Action of Azadirachtin
5.7.1. Studies using Insect Cell Lines
The analysis of direct cytological effects of azadirachtin
in whole insects, or even in isolated insect
tissues, is complicated by the multiplicity of physiological
consequences resulting from the compound
(see Section 5.6). This complexity has been
addressed by analyzing the pharmacology of azadirachtin
uptake and binding in cultured S. frugiperda
Sf9 cells (derived from pupal ovarian tissue) and
D. melanogaster Kc167 cells (derived from embryonic
cells). Such cells rapidly accumulate [ 3 H]azadirachtin
from culture medium and are enriched
with binding sites compared with many tissue
extracts derived from whole insects (Nisbet et al.,
1995, 1997; Mordue (Luntz) et al., 1999; Mordue
(Luntz), 2002; Robertson, 2004). The effects of
azadirachtin on the proliferation and cell cycle
events in these cells (Rembold and Annadurai,
1993; Salehzadeh et al., 2002, 2003; Robertson,
2004), accompanied by the above binding studies
have given detailed insights into the mode of action
of the compound.
Initial studies examining the effects of azadirachtin
on the proliferation of Sf9 cells used relatively
high concentrations (1 mM) in the culture medium
to produce profound effects on cell proliferation
(Rembold and Annadurai, 1993; Mordue (Luntz)
and Nisbet, 2000). Robertson (2004) has also shown
significant reductions in the number of viable cells
using the trypan blue exclusion assay within 48 h of
treatment with 1 mM azadirachtin for both Sf9 and
Kc167 cells. In addition to this effect on cell proliferation,
azadirachtin evoked a characteristic cytotoxic
effect on cultured cells. Reed and Majumdar (1998)
used azadirachtin concentrations of about 0.1 mM in
cell culture to demonstrate increases in cell volume,
blebbings, blisters, and holes in cell membranes resulting
in cytoplasmic extrusions. Since these studies
much more refined work has been carried out to discover
where in the cell azadirachtin is acting and at
what concentration.
5.7.2. Effects on Cell Cycle Events
The effects of azadirachtin in blocking cell division
have already been established in whole tissues such
as imaginal wing discs of E. varivestis (Schlüter,