Contents - Faperta

Molecular Techniques for Developing New Insecticide Molecules 495
Daborn et al., 2002; Ranson et al., 2002). Resistance mechanisms involving enhanced
detoxifi cation of insecticides and rendering the target insensitive to insecticides have
been detected earlier. One of the detoxifi cation mechanisms involving sequestration has
been reported in the case of resistance to organophosphates and carbamates in aphids
(Field, Devonshire, and Forde, 1988) and culicine mosquitoes (Raymond et al., 1998). The
other detoxifi cation mechanism, involving active degradation of the insecticides, has been
observed in two species of fl ies as a result of structural mutations in specifi c carboxylesterases
that converted them to kinetically ineffi cient but physiologically effi cient organophosphate
hydrolases (Newcomb et al., 1997; Campbell et al., 1998; Claudianos, Russel,
and Oakeshott, 1999). The third mechanism, in which the target molecule mutated in
such a way that it became insensitive to insecticides, has now been found in several
species involving different types of chemicals (Mutero et al., 1994; Williamson et al., 1996;
Vaughan, Rocheleau, and Ffrench-Constant, 1997; Ffrench-Constant et al., 2000; Martin
et al., 2000). The mutant target molecules included acetylcholinesterase for organophosphates,
g -aminobutyric acid (GABA) receptors for cyclodienes, and voltage-gated sodium
channels for the synthetic pyrethroids and dichlorodiphenyltrichloroethane (DDT).
The knock down resistance (kdr) gene in the housefl y, Musca domestica L., confers resistance
to rapid paralysis (knockdown) and lethal effects of DDT and pyrethroids. Flies with
the kdr trait exhibit reduced neuronal sensitivity to these compounds, which are known to
act at voltage-sensitive sodium channels of nerve membranes. The kdr trait is tightly linked
(within about 1 map unit) to the voltage-sensitive sodium channel gene segment exhibiting
the DNA sequence polymorphism. Resistance to pyrethroids has been well described
at the molecular level for several insect species. Consensus polymerase chain reaction
(PCR) primers that amplify a segment of the para-like sodium channel gene, which is
the molecular target of pyrethroids, have also been developed (Knipple et al., 1994). This
segment is known to carry pyrethroid resistance conferring point mutations (kdr and
superkdr) in Diptera and other insects, and although homologous, differed from published
genomic sequences of Blatella germanica (L.) and M. domestica for intron position and size.
The kdr mutation was found at a high frequency and could be associated to a low level of
pyrethroid resistance. In contrast, the superkdr mutation that confers high levels of insecticide
resistance in Diptera was not found in Frankliniella occidentalis (Perg.). However,
another point mutation close to the superkdr position was signifi cantly linked to pyrethroid
resistance in F. occidentalis. Combined occurrence of kdr and “thrips-superkdr ” allows for a
correct diagnosis of pyrethroid resistance in 94% of the individuals tested.
Comparative Genomics and Divergent Evolution of Detoxification Genes
One of the major advances in the study of insecticide resistance enabled by genomics has
been the cataloguing of relevant gene families. The lack of such information has been a
major constraint in studying resistance based on sequestration and degradation. Three
gene families that have been involved in insecticide detoxifi cation [cytochrome P450s,
carboxylesterases, and glutathione-S-transferases (GSTs)] have been fully sequenced in
Drosophila melanogaster Meigen and Anopheles gambiae (Giles) (Ranson et al., 2002). Most of
the P450s and GSTs are thought to have detoxifi cation or related digestive and/or metabolic
functions (Stevens et al., 2000; Tijet, Helvig, and Feyereisen, 2001), although many of
the esterases are also expected to have specialist nondetoxifi cation functions (Claudianos
et al., 2002). There is substantial scope for secondment of various members of these families
to resistance-related functions. The P450s seem to be the main resource for evolution
of resistance through enhanced detoxifi cation (Feyereisen, 1999). Although D. melanogaster