1011 J. Hemingway Insect Biochemistry and Molecular Biology 30 2000 1009–1015
other amplified variants of the same esterase loci, as they occur in
.80 of all characterized insecticide resistant strains. The local invasion of this amplicon into Culex
populations in southern France is well documented, Raymond et al., 1998. It was first found near Marseilles
airport and spread within a few years to all surrounding organophosphorus OP treated areas, despite the earlier
occurrence of other OP resistance mechanisms in this Culex population Raymond et al., 1998. The reason for
a selective advantage is not immediately apparent, as all the elevated esterases have similar affinities and turnover
rates for the different insecticides Karunaratne et al., 1993.
At least eight different esterase-containing amplicons have been recorded in Culex. One major difference
between amplicons is the presence of an aldehyde oxi- dase ao1 gene on the est
a2
1
est b2
1
amplicon. This is expressed in insects with this amplicon, but is found only
as a series of truncated 3 9 ao ends on the esta3 estb1
amplicons in other Culex strains Hemingway et al., 2000. The role of this amplified ao1 gene is not yet fully
characterized, although it is elevated in activity assays in resistant compared to susceptible insects, and interacts
with insecticides and herbicides containing aldehyde groups, hence a functional role is possible.
The est a and estb genes are the result of an ancient
gene duplication which appears to predate Culex speci- ation Hemingway and Karunaratne, 1998. The two
genes occur as single copies in a head to head arrange- ment 1.7 kb apart in the susceptible PelSS strain of C.
quinquefasciatus from Sri Lanka. In resistant insects with the est
a2
1
est b2
1
amplicon the intergenic spacer has been expanded to 2.7 kb with the insertion of two
large and one small indels Vaughan et al., 1997 com- pared to the susceptible PelSS spacer. The intergenic
spacer in other susceptible strains is variable in size, Guillemaud et al. 1996, 1999. The insertions in the
resistant spacer introduce a number of possible zeste regulatory
sequences into
the intergenic
spacer Hemingway et al., 1998. These elements, which affect
expression of multiple gene copies in Drosophila, may influence the levels of expression of the amplified ester-
ases Benson and Pirrotta, 1988. In contrast to the Culex amplified esterases, which are expressed in all life
stages, the E4 esterase gene of aphids can be switched off completely in revertant insects by methylation of the
gene. The pattern of methylation differs from many other organisms, where methylated genes are usually switched
off. In aphids E4-related sequences are highly methyl- ated at Msp1 sites in all resistant aphid clones, but not
in revertant clones, Field et al., 1989. Although the est
a2
1
and est b2
1
genes are present in a 1:1 stoichi- ometry, there is up to four times more Est
β 2
1
produced in the resistant insects, Paton et al., 2000. This differ-
ence in protein level is reflected in the expression pat- terns, although there is no direct link between activity
and amplification level in either resistant C. quinquefas- ciatus or C. tritaeniorhydchus, Paton et al., 2000. Clon-
ing of the intergenic spacer in both orientations upstream of a luciferase reporter gene has resulted in preliminary
characterization of the est
b2
1
promoter, Hemingway et al., 1998.
The est a2
1
promoter is inoperative when inserted at the same site. The difference in promoter strength may
reflect differences in tissue specific expression of the esterases, as changing the relative position of the puta-
tive est a2
1
promoter with respect to the luciferase reporter gene does not influence expression Hawkes and
Hemingway, [in preparation]. The amplified est a2
1
gene is expressed at a high level only in the malpighian tubules, cuticle, gut and salivary glands, Fig. 2, whilst
the expression pattern of the est b2
1
gene is as yet uncharacterized.
3. GST-based resistance
The glutathione S-transferases GSTs belong to a superfamily which currently has almost 100 sequences.
There are at least 25 groups families of GST-like pro- teins, with one well supported large clade containing
currently recognised mammalian, arthropod, helminth, nematode and mollusc GST classes Snyder and Maddi-
son, 1997. GSTs can produce resistance to a range of insecticides by conjugating reduced glutathione GSH
to the insecticide or its primary toxic metabolic products. The majority of reports involve organophosphate resist-
ance in houseflies Clark et al., 1986; Motoyama and Dauterman 1977, 1975, however, recent work on
recombinant Anopheles class I GST enzymes has shown that they recognize pyrethroids as either substrates or
inhibitors, Ranson et al., 1997; Prapanthadara et al., 1998, and there is now evidence that they are directly
involved in pyrethroid resistance in the planthopper N. lugens, Vontas, Small and Hemingway, [unpublished
data]. A subset of GSTs are also able to dehydrochlorin- ate insecticide such as DDT, in a reaction where GSH
acts as a co-factor rather than a conjugate Clark and Shamaan, 1984. This is probably the most common
DDT resistance mechanism in mosquitoes. Where conju- gation of primary metabolites occurs, the GST mech-
anism often acts as a secondary resistance mechanism in linkage disequilibrium with a monooxygenase or ester-
ase-based resistance mechanism, as in An. subpictus Hemingway et al., 1991.
The molecular basis of GST-based resistance is best understood in Musca domestica and the mosquitoes
Anopheles gambiae and Aedes aegypti. In all cases, upre- gulation of one or more GSTs in resistant insects appears
to be due to an, as yet, uncharacterized trans-acting regu- lator Grant and Hammock, 1992. Active research pro-
grammes are under way in a number of laboratories to
1012 J. Hemingway Insect Biochemistry and Molecular Biology 30 2000 1009–1015
identify and characterize these regulators using a pos- itional cloning approach which has already identified
crude chromosomal locations, which should contain these regulatory genes Ranson et al., 1999.
Insect GSTs are currently classified into two groups, class I and class II GSTs. This classification is almost
certainly over simplified. Class I GSTs are most closely
Fig. 2. High resolution electron micrograph
× 25,000 of the est
a2
1
expression visualized by gold labelling in: A the salivary gland of insecticide resistant 4th instar larval Culex quinquefasciatus, and, B
the cuticle of resistant 4th instar larvae compared to the lack of staining in C, the cuticle of insecticide susceptible larvae. Salivary glands
were dissected out of individual larvae and sectioned. The cuticle was visualised in cross-sections of whole larvae. Fresh salivary gland was
lightly fixed in a glutaraldehyde:formaldehyde mixture for 30 minutes at 4
° C and was prepared using the Tokuyasu protocol for immuno-
electronmicroscopy. After fixation, the material was infused with 2.3 M sucrose and then vitrified using liquid N
2
. Sections were cut at 2100
° C using a Reichert Ultracut E fitted with a FC4 Cryochamber
and were thawed onto 2 M sucrose solution. After treatment with pri- mary antibody Ab2; diluted 1:200, and secondary rabbit IgG conju-
gated to 10 nm gold particles, the sections were embedded on their grids using a methyl cellulose:uranyl acetate mix before examination
in the electron microscope. For micrographs B and C after sec- tioning, the material was lightly fixed in a glutaraldehyde:paraformal-
dehyde mixture for 30 minutes at 4
° C, and after rapid dehydration in
70 alcohol was embedded in Hard Grade LR White resin. Sections nominally 60 nm thick were cut using a Reichert Ultracut E ultramicro-
tome and placed onto copper EM grids. These were treated with pri- mary antibody Ab2 made against purified Culex esterase at 1:200
dilution and then with rabbit IgG conjugated to 10 nm colloidal gold. Immunolabelling was followed by routine uranyl acetate and lead cit-
rate staining. Sections were examined and photographed using a JEOL 1210 TEM. Labelling shows as small intense black dots.
related at the amino acid level to mammalian theta class GSTs, while class II GSTs are related to the pi class see
Fig. 3, this relationship between insect and mammalian classes does not extend to their substrate specificities.
The complexity of the class I GSTs in different insect species are highly variable. In Drosophila melanogaster,
this class comprises six divergently organized intronless genes on a contiguous stretch of chromosome Toung et
al., 1993. In Aedes aegypti, metabolically-based DDT resistance is associated primarily with an increase in
expression levels of a single GST class II enzyme Grant and Hammock, 1992; Grant and Matsumura, 1989. The
resistance gene appears to be a trans-acting regulator that affects expression of this GST Grant and Ham-
mock, 1992. In contrast, in An. gambiae, biochemical analyses indicate a large number of GSTs are involved in
resistance. The subsets of GSTs involved varied between larvae and adults Prapanthadara et al. 1993, 1995.
Complex but distinct subsets of these were upregulated in resistant compared to susceptible mosquitoes at both
life stages. In this species, within a class I GST cluster, there are three genes in divergent orientations. There is
a single intronless GST, a further GST with a common 5
9 end, which can be alternately spliced to four different 3
9 ends Ranson, 1996; Ranson et al. 1997, 1998, and a gene split by two introns Roberts and Hemingway,
unpublished data, see Fig. 4. Further variability occurs within An. gambiae populations through multiple allelic
variants within the 3 9 ends of the spliced GSTs and the
intronless gene Ranson et al., 1998. The 5 9 end of the
gene controls GST binding, while the 3 9 end determines
substrate specificity.
1013 J. Hemingway Insect Biochemistry and Molecular Biology 30 2000 1009–1015
Fig. 3. Phylogenetic tree of the insect glutathione S-transferases with selected mammalian and parasite GST sequences. Bootstrap values are
given for the insect GST relationships.
Fig. 4. Diagrammatic representation of the Anopheles gambiae Class I GST gene cluster. Six different GST transcripts are produced from the
genes in both resistant and susceptible insects. Four of the transcripts originate from alternate splicing of the aggst1 α
gene.
The class I GSTs cloned to date still represent only a tiny subset of the GST variation seen biochemically in
An. gambiae which suggests that there are still further insect GST classes for which we do not have molecular
data. Screening of an An. gambiae BAC library has con- firmed this and details of new GSTs should be published
shortly Ranson and Collins [personal communication].
There are a number of possible regulatory elements upstream of the cloned GSTs, but the regulatory mech-
anism producing resistance still needs to be charac- terized. A positional cloning programme, using the An.
gambiae microsatellite markers, have identified a QTL in which the probable trans-acting regulator of An. gam-
biae should reside. Further microsatellite loci are being characterized in the regions of this QTL for fine scale
mapping, which should identify possible candidate regu- latory genes in the near future Ranson et al., 1999.
1014 J. Hemingway Insect Biochemistry and Molecular Biology 30 2000 1009–1015
4. Conclusions
