Insect Control: Biological and Synthetic Agents - Index of

290 8: Mosquitocidal B. sphaericus: Toxins, Genetics, Mode of Action, Use, and Resistance Mechanisms
8.3.1.1. Cytopathology and physiological
effects Bti toxins completely breakdown the larval
midgut epithelium, whereas B. sphaericus does not.
Nevertheless, midgut alterations start within 15 min
of feeding on B. sphaericus spore/crystal complex
(Davidson, 1981; Karch and Coz, 1983; Charles,
1987; Singh and Gill, 1988). There is no difference
between midgut damage in C. pipiens after ingestion
of spore/crystals from strains 1593 or 2297
(Davidson, 1981; Charles, 1987; Singh and Gill,
1988), even though these two strains belong to
different Bin types. In contrast, symptoms of intoxication
are not identical in different mosquito species.
Large vacuoles (and/or cytolysosomes) appear
in C. pipiens midgut cells, whereas large areas of
low electron density appear in Anopheles stephensi
midgut cells (compare Figure 3c and d with the
control, Figure 3b). Mitochondrial swelling is a
general feature described for C. pipiens and A. stephensi,
as well as for A. aegypti, when intoxicated
with a very high dose of spore/crystals (Charles,
1987). The midgut cells, especially cells of the posterior
stomach and the gastric caecae, are the most
severely damaged by the toxin, and Singh and Gill
(Singh and Gill, 1988) also reported late damage
to neural tissue and skeletal muscles.
Ultrastructural effects have also been observed in
cultured cells of C. quinquefasciatus within few
minutes of treatment with soluble and activated
B. sphaericus toxin. These alterations consisted
mainly in the swelling of mitochondria cristae
and endoplasmic reticula, followed by the enlargement
of vacuoles and the condensation of the
mitochondrial matrix (Davidson et al., 1987).
The physiological effects of B. sphaericus crystal
toxin have been very poorly documented. The
oxygen uptake of mitochondria isolated from
B. sphaericus-treated C. quinquefasciatus larvae is
inhibited, as is the activity of larval choline acetyl
transferase (Narasu and Gopinathan, 1988). In addition,
oxygen uptake by mitochondria isolated
from rat liver is reduced by the B. sphaericus toxin
(Narasu and Gopinathan, 1988).
8.3.1.2. Binding to a specific receptor in the brush
border membrane As the differences in susceptibility
between mosquito species do not seem to be due
to the ability to solubilize and/or to activate the
binary toxin, the variation in susceptibility is presumably
due to cellular differences. Indeed,
fluorescently-labeled toxin binds to the gastric
caecae and the posterior stomach only in very susceptible
Culex species. BinB does not bind to the gut
of Aedes aegypti, whereas BinA weakly and nonspecifically
binds in this species, and no regionalized
binding occurs for Anopheles (Davidson, 1988;
Davidson, 1989; Davidson and Yousten, 1990;
Oei et al., 1992). Furthermore, in C. quinquefasciatus,
only BinB binds to the caecae and posterior
stomach, and the binding of BinA appears to be
conditioned by the binding of the BinB (Figure 4b),
whereas BinA alone binds nonspecifically throughout
the midgut (Figure 4a). This indicates that only
one of the two components of the crystal toxin
(putatively BinB) specifically binds to the midgut
of susceptible larvae. The hypothesis that the
N-terminal region of BinB is involved in regional
binding of this protein in the larval midgut, and
that the C-terminal region of BinB, as well as both
the N- and C-terminal regions of BinA are involved
in the interaction of the two components leading
to the binding of BinA in the same regions as BinB,
was supported by early in vivo binding studies using
nontoxic deletion mutants of the crystal toxin (Oei
et al., 1992). The same authors also showed that the
Bin toxin is only internalized when both components
are present. However, further intracellular
investigations are required to determine whether
one or both components are internalized.
In vitro binding assays, using 125 I-labeled activated
crystal toxin and brush border membrane
fractions (BBMFs) isolated from susceptible or nonsusceptible
mosquito larvae, confirmed this hypothesis.
Indeed, direct binding experiments with
C. pipiens BBMFs indicated that the toxin binds
to a single class of specific receptor (Figure 5, left;
Nielsen-LeRoux and Charles, 1992). No significant
specific binding was detected with BBMFs from
A. aegypti (Figure 5, right), which is consistent
Figure 4 (a) Midgut of C. quinquefasciatus larvae only fed with
fluorescently-labeled BinA. BinA weakly binds over the entire
midgut. (b) Larvae fed with fluorescently-labeled BinA and
unlabeled BinB; BinA binding is restricted to the gastric caecae
and posterior stomach. am, anterior stomach; gc, gastric caeca;
pm, posterior stomach. (Micrographs kindly provided by Dr
C. Berry, Department of Biochemistry, Cardiff.)