Insect Control: Biological and Synthetic Agents - Index of

20 1: Pyrethroids
early 1950s in houseflies (Busvine, 1951). It causes a
loss of sensitivity to DDT and its analogs, and
to pyrethrins and pyrethroids, which all owe their
activity to interaction with the para-type voltagegated
sodium channel in nerve membranes. This
loss of activity is characterized by a reduction in
the binding of these insecticides to the sodium channel
(Pauron et al., 1989). An enhanced form of this
resistance termed super-kdr has also been characterized
in houseflies (Sawicki, 1978). Both the kdr and
super-kdr traits were mapped to chromosome 3 and
found to occupy the same allele, the para-type sodium
channel. This has been confirmed by molecular
cloning studies of these channels in kdr and superkdr
houseflies (Williamson et al., 1996) and kdr
B. germanica (Miyazaki et al., 1996; Dong, 1997).
The super-kdr resistance in houseflies is due to a
methionine to threonine (M918T) point mutation
in the gene encoding the para sodium channel.
Both mutations were located with domain II of the
ion channel. The L1014F mutation in IIS6 was
found in both housefly and cockroach strains, and
confers kdr resistance. To date, the M918T mutation
has not been detected as a single substitution in any
housefly strain, but only occurs in conjunction with
L1014F. Mammals are intrinsically much less susceptible
to pyrethroids and DDT. Significantly,
mammalian neuronal sodium channels have an isoleucine
rather than methionine in the position (874)
that corresponds to the housefly super-kdr site (918).
Site-directed mutation of this residue to methionine
gives rise to a channel with >100x increased sensitivity
to deltamethrin, suggesting that differential
pyrethroid sensitivity between mammals and insects
may be due in part to structural differences between
the mammalian and insect sodium channels (Vais
et al., 2000). In some insect species, the existence
of kdr-type target-site resistance has often been
masked by efficient metabolic resistance mechanisms;
an example is M. persicae, in which resistance-associated
esterase is responsible for much of
the reduced sensitivity towards organophosphates,
carbamates, and pyrethroids. Any inference that
target-site resistance might also be a factor has usually
been based on the sensitivity of the insects in
the presence of synergists such as PBO or DEF S,S,S,tributyl
phosphorotrithiolate, and the assumption
that any residual decreased sensitivity is due to
kdr-type target-site resistance.
Use of degenerate DNA primers for the para-type
sodium channel and sequencing of the gene have
resulted in the unequivocal identification of resistance-inducing
mutations in the trans-membrane
domain II (Martinez-Torres et al., 1999b) of
the channel in M. persicae, in which a leucine to
phenylalanine (L1014F) mutation associated with
kdr was identified. Insects containing this mutation
could also be identified by the use of a discriminating
dose of DDT, as DDT is unaffected by the enhancedesterase
mechanisms also present in most of the
aphid clones. Indeed, of 58 aphid clones analysed
for both kdr- and esterase-based mechanisms, only
four contained an esterase (E4) mutation and not
kdr. Estimates for resistance factors in aphids containing
both mechanisms are 150–540-fold,
in comparison to 3–4-fold (FE4 esterase alone) or
35-fold for kdr alone. Consequently, these dualresistance
mechanisms afford a level of decreased
sensitivity whereby insects become totally immune
to field dosages of pyrethroids. Similar kdr mutations
have also been detected in cockroaches
(Miyazaki et al., 1996; Dong, 1997), H. irritans
(Guerrero et al., 1997), P. xylostella (Schuler et al.,
1998), and An. gambiae (Martinez-Torres et al.,
1998). In a pyrethroid-resistant strain of the tobacco
budworm H. virescens, the same locus was mutated
to histidine rather than phenylalanine (Park and
Taylor, 1997)
As with metabolic resistance mechanisms, it is
important to establish methods that can identify
sodium-channel kdr-type mechanisms in single
insects so that it is possible to adjust insect-control
methods. The kdr-mutation of nerve insensitivity
was originally identified by electrophysiology, and
this method still remains a fundamental way of
confirming nerve insensitivity. However, it is a
specialized and rather cumbersome technique that
is out of the question when attempting to test large
numbers of an agricultural pest species. The DDT
bioassay using a discriminating dose remains a useful
method but may not completely discriminate
between homozygous and heterozygous individuals.
The most useful technique has been direct diagnosis
of the mutation(s) based on PCR amplification and
sequencing. The identification of the L1014F mutation
in knockdown-resistant housefly strains has led
to the development of several diagnostic assays for
its occurrence in other species, including H. irritans
(Guerrero et al., 1997), the mosquitoes An. gambiae
(Martinez-Torres et al., 1998), and Culex pipiens
(Martinez-Torres et al., 1999a), as well as M. persicae.
However, this technique will only identify
known mutations and the test designed to detect
L1014F in An. gambiae (Ranson et al., 2000) did
not detect the additional L1014S.
The molecular biology of knockdown resistance
to pyrethroids has been reviewed (Soderlund and
Knipple, 2003). Sequencing of the para-sodium
channel gene from several arthropod species has
led to the discovery of a number of amino-acid