Insect Control: Biological and Synthetic Agents - Index of

8: Mosquitocidal B. sphaericus: Toxins, Genetics, Mode of Action, Use, and Resistance Mechanisms 301
unpublished data). Further studies are required
to evaluate whether strain IAB-59 is of commercial
value as an alternative to B. sphaericus strains 1593,
2362, 101-B, and C3-41 (the only strains currently
produced for use in the field). This depends on its
potency and field performance. Additionally, several
groups are currently trying to identify the genes
encoding the additional toxic compounds from
strains IAB-59 and LP1-G.
The fact that the Bin toxin behaves as a unique
moiety during the binding step indicates that the
probability of selecting resistance to B. sphaericus
is much higher than for Bti, which contains at least
four proteins that potentially act on different target
molecules. Despite reports of laboratory and field
resistance, the use of B. sphaericus for the control
of mosquito larvae is still of interest. Nevertheless, it
is important to collect information about baseline
susceptibility before starting treatment. However, as
resistance to B. sphaericus is recessive, it is only possible
to detect resistance when it has already become
homozygous and therefore difficult to handle. Thus, it
may be possible to monitor the risk of resistance development
in a given area by use of molecular nucleotide
probes based on the mutation of resistance genes.
However, resistance to B. sphaericus seems to be very
complex and different probes would be needed to
cover all possible mutations, only one of which has
been identified so far (Darboux et al., 2002).
Both from a practical and an ecological point of
view, the best and most direct way to overcome
and to manage B. sphaericus resistance is of course
Bti, which is already commercialised. Fortunately,
all Culex populations reported to display field resistance
to B. sphaericus have been tested for
susceptibility to Bti. No cross-resistance has been
observed. There is even some evidence for increased
susceptibility in some colonies (Rao et al., 1995;
Silva-Filha et al., 1995; Yuan et al., 2000). This
was expected, because the multi-toxin complex of
this bacterium does not share any receptor-binding
sites with the B. sphaericus binary toxin (Nielsen-
LeRoux and Charles, 1992). In several situations, it
is recommended that Bti be used in rotation with
B. sphaericus (Regis and Nielsen-LeRoux, 2000;
Yuan et al., 2000). Recent results obtained in
Thailand have suggested that a mixture of Bti and
B. sphaericus may help reduce the risk of appearance
of B. sphaericus resistance while preserving
the superior field performance of B. sphaericus
compared to Bti, particularly in breeding sites rich
in organic matter (Mulla et al., 2003).
Alternatives to natural Bti and B. sphaericus
strains include genetically modified organisms, as
combining different toxins binding to different
receptors in the same organism is also a good way
of preventing resistance. In addition to the Bin toxins,
other toxins from either B. sphaericus or other
insecticidal microorganisms could be produced. For
example, the expression of Mtx1 genes in B. sphaericus
during sporulation, i.e., under the control of a
sporulation promoter, could allow the diversification
of toxins. Similarly, Bti mosquitocidal toxin
genes could be combined with B. sphaericus genes;
some groups have already attempted this, for example
by introducing the Bti genes encoding Cry4B,
Cry4D or Cry11A into toxic B. sphaericus strains
(Trisrisook et al., 1990; Bar et al., 1991; Poncet
et al., 1994; Poncet et al., 1997). Conversely,
B. sphaericus crystal toxin genes have been successfully
introduced into toxic Bti (Bourgouin et al.,
1990). Although these studies did not demonstrate
any increase in toxicity against Anopheles and
Culex, such recombinants may delay the emergence
of resistant insects.
Several attempts have been made to introduce or
to combine toxin genes from Bti, B. thuringiensis
serovar jegathesan (Bt jeg) and serovar medellin
(Bt med), and to evaluate their activity towards
both susceptible and B. sphaericus-resistant colonies.
The introduction of Cry11A from Bti and
Cry11Ba from Bt jeg partially restored the susceptibility
of resistant colonies (Poncet et al., 1997;
Servant et al., 1999). Nonspecific cytolytic toxins
(Cyt1A and Cyt1B) also partially restored its toxicity
towards two resistant colonies (Thiéry et al.,
1998; Wirth et al., 2000a). A Bti strain harboring
genes encoding the binary toxin as well as Cyt1 and
Cry11B was more toxic than unmodified Bti IPS-82
(Park et al., 2003). Several groups have also
attempted to introduce and to overproduce entomopathogenic
toxins into organisms living in the
environment and serving as natural foods for mosquitoes,
such as cyanobacteria (Liu et al., 1996b), or
to stably integrate the toxins encoding these genes
into the chromosomes of other bacteria such as
Enterobacter amnigenus (Tanapongpipat et al.,
2003). However, given our current knowledge of
B. sphaericus resistance, the expression of Bin
toxin as the only active compound should be
avoided. No recombinant strains have yet proved
to have a better field performance than the natural
strains, and no products based on recombinant
strains have yet been registered for use in mosquito
control programs. Therefore there is a continuing
need to identify new strains with novel modes of
action against the principal mosquito vectors of
human diseases.