Insect Control: Biological and Synthetic Agents - Index of

A1.4. Resistance
The increasing number of reports of pyrethroid
resistance in the field has fuelled further studies
to determine the molecular basis of resistance, and
recent progress in this area is summarized in
Table A1. Several cytochrome P450s that are overexpressed
in insecticide-resistant strains and are able
to metabolize pyrethroids have been identified. For
example, the Anopheles gambiae enzyme, CYP6P3,
is upregulated in pyrethroid-resistant populations
from Ghana and Benin and it metabolizes both
a-cyano (type 2) and non-a-cyano (type 1) pyrethroids
(Muller et al., 2008). Another CYP6 enzyme
from the mosquito An. minimus has also been demonstrated
to have activity against deltamethrin, supporting
its likely role in resistance (Boonsuepsakul
et al., 2008). A number of P450s from Helicoverpa
armigera have been implicated in pyrethroid resistance
by metabolism studies (Table A1), although
genetic mapping has suggested that the in vitro ability
of certain P450s (such as the CYP6B7 and CYP6B6)
to metabolize pyrethroids may not be sufficient evidence
to prove their role in resistance (Grubor and
Heckel, 2007). The application of RNA silencing techniques
has demonstrated the importance of CYP6BG1
from the diamondback moth, Plutella xylostella,
in conferring permethrin resistance (Bautista et al.,
2009). Recently, the gene duplication events in
An. funestus as a possible contributing mechanism
fortheincreasedP450activityinpyrethroidresistance
populations were identified in this enzyme family in
insects (Wondji et al., 2009).
The number of insect and mite species documented
as containing mutations in the pyrethroid target
site, the voltage-gated sodium channel of nerve
membranes, continues to grow (see Table A1).
Many of these are clustered within localized ‘‘hotspots,’’
including the domain IIS4-S5 linker, IIS5 and
IIS6 helices and the corresponding regions of
domains I and III (reviewed in Davies et al., 2007),
and have been instrumental to identify a clearly
defined binding site for these compounds at the
sodium channel pore (see Section 1.5). The functionality
of many of these mutations has now been
confirmed by analysis of their properties in Xenopus
oocyte-expressed insect channels, and some of the
more commonly identified mutations at residues,
such as M918, T929, L1014 and F1538, confer
insensitivity across a range of pyrethroid structures.
The resistance mechanism is corroborated when the
same, or similar, mutations are found to occur in a
new species; for example, the recent identification of
F1538I in the spider mite Tetranychus urticae
(Tsagkarakou et al., 2009). Other mutation sites
A1: Addendum 31
have been identified that are clearly linked to the
resistance phenotype but have not been functionally
characterized by in vitro assay; for example, V1016I
in Aedes aegypti (Saavedra-Rodriguez et al., 2007).
A note of caution, however, is that, not all reported
mutations have been so clearly linked to pyrethroid
resistance and some may represent natural polymorphisms
within the sodium channel sequence.
Also, secondary processing events known to generate
developmental and/or tissue-specific diversity of mature
sodium channel isoforms may also affect the level
of resistance imparted by a particular mutation; for
example, the expression of alternatively spliced
sodium channel transcripts in resistant diamondback
moth populations produced distinct channels with
different sensitivities to pyrethroids (Sonoda et al.,
2008). Finally, in populations with multiple resistance
mechanisms, the relative contribution of each mutation
to the resistance phenotype is often poorly defined.
This has been explored in Culex mosquitoes
and revealed a complex interplay between metabolic
and target site resistance (Hardstone et al., 2009).
A1.5. Pyrethroid–Sodium Channel
Interactions
One area in which there has been significant progress
over the past 5 years is in the development
of a detailed molecular model showing a putative
binding site for pyrethroids at the insect sodium
channel. This was made possible by publication of a
series of high-resolution crystal structures for closely
related potassium channels, including the eukaryotic
voltage-gated K + channel Kv1.2 (Long et al., 2005),
that provided a structural template on which to model
the insect (housefly) sodium channel sequence
(O’Reilly et al., 2006). Close examination of the
pore region of this model revealed a long, narrow
cavity between the IIS4-S5 linker, IIS5 helix and
IIIS6 helix that could accommodate a range of pyrethroid
structures as well as DDT (see Figure A1).
Several known features of pyrethroid mode of action
and resistance are supported by this model (O’Reilly
et al., 2006), including (1) a hydrophobic cavity accessible
to lipid-soluble pyrethroids, (2) the observation
that the binding site is formed by movement of
the IIS4-S5 linker towards the other two helices during
channel activation, which is consistent with
electrophysiological evidence that pyrethroids bind
preferentially to the open state of the channel, (3)
that this high-affinity binding would stabilize the
open state to slow channel inactivation, consistent
with the pyrethroid-induced tail currents seen following
membrane repolarization, (4) the proposed binding
site includes many known mutation sites in the