Insect Control: Biological and Synthetic Agents - Index of

18 1: Pyrethroids
because there is no such thing as a specific inhibitor
and some compounds thought to be specific inhibitors
of cytochromes P450 (e.g., piperonyl butoxide)
may also inhibit esterases (Gunning et al.,
1998b; Moores et al., 2002).
Studies have included both in vivo work on
whole insects or their tissues and in vitro studies
with isolated enzymes. In resistant populations
of many insect species, the mechanisms are most
frequently due to both enhanced esterase and cytochrome
P450 levels, so that dissection of the proportions
of the different mechanisms is difficult.
However, evidence that resistance ratios are reduced
or abolished in the presence of reliably specific
inhibitors such as organophosphates (Gunning
et al., 1999; Corbel et al., 2003) can be taken as a
good indication that enhanced esterase levels are
responsible for metabolic resistance.
1.5.1.1.4. Cytochrome P450 monooxygenases
These are a class of Phase I detoxification enzymes
that catalyse various NADPH- and ATP-dependent
oxidations, dealkylations, and dehydrogenations.
Both microsomal and mitochondrial forms occur
in insects. They are probably responsible for the
most frequent type of metabolism-based insecticide
resistance (Oppenoorth, 1985; Mullin and Scott,
1992; Scott and Wen, 2001). They are also a major
mechanism for pyrethroid catabolism (Tomita and
Scott, 1995). Their occurrence and importance
in insect xenobiotic metabolism has been reviewed
by Scott and Wen (2001). The super-family of
cytochrome P450 genes has probably evolved by
gene duplication and adaptive diversification, and
comprises 86 functional genes in D. melanogaster.
The large number of substrates metabolized by
P450s is due both to the multiple isoforms and to
the fact that each P450 may have several substrates
(Rendic and DiCarlo, 1997). Because these enzymes
may have overlapping substrate specificities, it is
difficult to ascribe the function to individual P450
enzymes. In insects, although the importance of
oxygenases in the metabolism of many substrates is
known, the particular P450 isoforms involved have
rarely been identified.
Several P450 iso-enzymes have been isolated or
expressed from insect sources. Regarding pyrethroid
metabolism, the best-characterized P450 isoform is
CYP6D1. This was originally purified from a strain
of highly resistant (ca. 5000) houseflies designated
‘‘Learn pyrethroid resistant’’ (LPR) selected by
the continuous usage of permethrin to control flies
in a New York State dairy. A reduced-penetration
mechanism and kdr were also present in the strain.
CYP6D1 has been purified (Wheelock and Scott,
1989) and sequenced via the use of degenerate primers
derived from known protein sequences and
PCR amplification (Tomita and Scott, 1995). Overproduction
of this P450 isozyme was found to be the
major mechanism of deltamethrin detoxification in
microsomes derived from the LPR flies (Wheelock
and Scott, 1992). The enzyme requires cytochrome b5
as a co-factor and is specific in its action, because
only the 4 0 -hydroxy metabolite was produced from
cypermethrin (Zhang and Scott, 1996). CYP6D1
was found to be the major and possibly the only
P450 isoform responsible for pyrethroid metabolism
in this strain of houseflies; consequently, the
resistance ratios are very much less for pyrethroids
such as fenfluthrin that do not have the 3-phenoxybenzyl
group (Scott and Georghiou, 1986). The
same mechanism was found to be responsible for
PBO suppressible resistance to permethrin from a
Georgia poultry farm in the USA (Kasai and Scott,
2000). In both these housefly strains, the mechanism
was due to an increased (ca. 10) transcription of
the gene, leading to increased levels of CYP6D1
mRNA and higher levels of the enzyme. CYP6D1
is expressed in the insect nervous system and has
been shown to protect the tissue from the effects of
cypermethrin (Korytko and Scott, 1998). Clearly,
from the metabolic specificity of CYP6D1, other
isoforms of cytochromes P450 must also be implicated
in pyrethroid metabolism, although which
reactions are catalyzed by which isoform has yet to
be determined.
It is characteristic of monooxygenases that they
are inducible within an individual animal. The use
of phenobarbitone to induce monooxygenase activity
in rat liver is well known, and many other agents
are capable of transiently up-regulating cytochromes
P450. Phytophagous insects are exposed
to many plant xenobiotics, for example monoterpenes
which also induce P450 production. Such
induction of P450s may incidentally induce an isoform
also capable of metabolizing pyrethroids. For
example, feeding larvae of H. armigera on mint
(Mentha piperita) leaves induced a 4 resistance to
pyrethroids compared with those fed on a semidefined
diet (Hoque, 1984; Terriere, 1984; Schuler,
1996; Scott et al., 1998). CYP6B2 mRNA, a P450
isoform also implicated in pyrethroid resistance,
is inducible by peppermint oil and specifically apinene
in larvae of H. armigera (Ranasinghe et al.,
1997). This induction was rapid (ca. 4 h) and disappeared
within a similar period of removing the
stimulus. Clearly, the mechanism for the transient
induction of P450s (Ramana, 1998) is different
from the situation with the LPR houseflies, in which
CYPD1 is permanently up-regulated (Liu and Scott,