Insect Control: Biological and Synthetic Agents - Index of

8: Mosquitocidal B. sphaericus: Toxins, Genetics, Mode of Action, Use, and Resistance Mechanisms 297
one, the stable low level resistant JRMM-R
(Rodcharoen and Mulla, 1994; Rodcharoen and
Mulla, 1997). In the field, resistance appears to
be unstable in the absence of selection pressure
because, as soon after B. sphaericus treatment is
interrupted, the exposed C. quinquefasciatus populations
become susceptible to B. sphaericus again
(Silva-Filha and Regis, 1997; Yuan et al., 2000).
8.5.2.1. Molecular basis of laboratory-selected
resistance As described above, the binding of
BinB to the a-glucosidase (Cpm1) receptor at
the apical membrane of midgut cells is essential
for larvicidal activity. Thus, when resistance to
B. sphaericus was first reported, the mechanisms
of resistance were analyzed by comparing the
kinetics of Bin receptor binding in the midguts of
GEO and susceptible C. quinquefasciatus larvae
(CpqS). As expected, the binding characteristics of
125 I-Bin to CpqS BBMFs were very similar to those
previously reported for C. pipiens membranes
(Nielsen-LeRoux and Charles, 1992). In contrast,
Bin cannot specifically bind BBMFs from GEO mosquitoes
(Nielsen-LeRoux et al., 1995). In addition,
when assayed on BBMFs prepared from F1 (CpqS
X GEO) larvae, 125 I-Bin bound to a single class
of receptor, which is consistent with the suspected
recessive nature of the resistance trait. Analysis
of binding data with BBMFs from the backcross
(BC) progeny showed that the binding sites are not
saturated in the range of toxin concentrations used,
and that the total amount of bound toxin is much
lower than for the susceptible parental strain.
LIGAND analysis showed that the experimental
data obtained with the BC progeny fit a two-site
model better than a one-site model. This possible
existence of two classes of binding sites in the BC
population is suggestive of genetic heterogeneity.
The next step was to determine whether this
receptor was absent from the GEO mosquito
midgut or whether it was present in a form that
could no longer recognize the Bin toxin. In Northern
blot experiments, total RNA extracted from GEO
midguts was probed with a cpm1 DNA fragment.
The 2-kb transcript previously identified in IP mosquitoes
was present in similar amounts in GEO
mosquitoes (Darboux and Pauron, unpublished
data). In situ hybridization experiments confirmed
that cpm1 transcripts are equally distributed both
quantitatively and qualitatively in CpqS and GEO
midguts. These transcripts were found in the
regions that had been previously identified as
Cpm1 reservoirs, i.e., cardia cells, the gastric caecae
and the posterior midgut (Darboux et al., 2002).
Nevertheless, the amount of Cpm1 protein differed
completely in the two populations. Firstly,
immunolocalization performed on the same type
of cryosections as the in situ hybridizations failed
to detect Cpm1 in any of the above-mentioned
structures or anywhere else in the larval midgut.
Secondly, Cpm1 was not detected in BBMFs from
GEO mosquitoes by Western blotting (Darboux et al.,
2002). Taken together, these results suggest that
the sequence encoding Cpm1 is altered in GEO.
Analysis of the full cDNA sequence of cpm1GEO
confirmed this hypothesis. The cpm1GEO sequence
contains seven nonsilent mutations compared to
cpm1 IP. Six of these mutations result in amino acid
substitutions in the protein itself (Ala95Asp;
Lys115Met; Glu178Thr; Asp230His; Asn265Asp;
Leu486Met), and the seventh introduces a termination
signal in the hydrophobic tail of the
protein (Leu569Stop). To determine which of these
mutations were involved in the resistance mechanism,
Sf9 cells were transfected with various constructs
corresponding to natural or chimeric forms of
Cpm1. Western blotting, binding experiments and
enzymatic assays were performed on the expression
products of each construct. The Cpm1GEO form
was unable to link to the plasma membranes of
Sf9 cells (Figure 7), but accumulated in the extracellular
medium, which explains why no signal
could be detected both in BBMF Western blotting
and by immunolocalization in midgut sections.
Concomitantly, no binding activity was detected on
membranes prepared from Sf9/Cpm1GEO cells, which
confirms the previous results of in vitro binding of
125 I-Bin toxin to BBMFGEO (Darboux et al., 2002).
Figure 7 Ectopic expression of Cpm1. Sf9 cells were transfected
with the cpm1IP sequence (Sf9-S) or the cpm1GEO one
(Sf9-GEO). The anti-Cpm1 antibody gives a signal only on the
plasma membrane of the Sf9-S cells (Castella and Pauron,
unpublished data).