Nicola J. Hawkes

Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene.

Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth‐instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S‐transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock‐down resistant (kdr)‐type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage‐gated sodium channel gene was obtained by RT‐PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains.

Can multiple-copy sequences of prey DNA be detected amongst the gut contents of invertebrate predators?

The first experiments to clearly demonstrate that DNA techniques might be used to detect predator–prey interactions between arthropods are reported. The accurate modelling of such interactions has depended until now upon a mixture of laboratory experiments, population monitoring and biochemical tests. The latter involve gut‐content analyses, and have most recently depended upon the development of prey‐specific monoclonal antibodies. Although these are excellent for detecting predation on a target prey, they are impractical for analysing the prey range of a particular predator. Molecular detection depends upon the ability of DNA to resist digestion in the predator gut and of the polymerase chain reaction (PCR) to amplify prey‐specific DNA from semidigested material. As a first step, experiments using carabid beetles, Pterostichus cupreus L., as predators and mosquitoes as prey are reported. The target sequences were fully characterized multiple‐copy esterase genes from two laboratory strains of Culex quinquefasciatus Say. Although DNA was extracted from homogenates of whole beetles (minus appendages), a 146 bp product could be amplified from both mosquito strains digested in the beetle gut for 28 h. The larger, 263 bp product was detectable for 28 h in one mosquito strain, but could not be amplified after 5 h from the other. Whether the beetles had eaten one mosquito or six, digested for zero or 28 h, the prey were equally detectable. Having demonstrated that shorter, multiple‐copy sequences survive digestion for a considerable period in the gut of a predator, the opportunity exists to develop new detection systems for studying predation in the field.

Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera oleae

A 2.2‐kb full length cDNA containing an ORF encoding a putative acetylcholinesterase (AChE) precursor of 673 amino acid residues was obtained by a combined degenerate PCR and RACE strategy from an organophosphate‐susceptible Bactrocera oleae strain. A comparison of cDNA sequences of individual insects from susceptible and resistant strains, coupled with an enzyme inhibition assay with omethoate, indicated a novel glycine‐serine substitution (G488S), at an amino acid residue which is highly conserved across species (G396 of Torpedocalifornica AChE), as a likely cause of AChE insensitivity. This mutation was also associated with a 35–40% reduction in AChE catalytic efficiency. The I199V substitution, which confers low levels of resistance in Drosophila, was also present in B. oleae (I214V) and in combination with G488S produced up to a 16‐fold decrease in insecticide sensitivity. This is the first agricultural pest where resistance has been associated with an alteration in AChE, which arises from point mutations located within the active site gorge of the enzyme.

Characterization of the promoters of Epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress.

Epsilon class GSTs (glutathione transferases) are expressed at higher levels in Anopheles gambiae mosquitoes that are resistant to DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] than in insecticide-susceptible individuals. At least one of the eight Epsilon GSTs in this species, GSTe2, efficiently metabolizes DDT to DDE [1,1-dichloro-2,2-bis-(p-chlorophenyl)ethane]. In the present study, we investigated the factors regulating expression of this class of GSTs. The activity of the promoter regions of GSTe2 and GSTe3 were compared between resistant and susceptible strains by transfecting recombinant reporter constructs into an A. gambiae cell line. The GSTe2 promoter from the resistant strain exhibited 2.8-fold higher activity than that of the susceptible strain. Six polymorphic sites were identified in the 352 bp sequence immediately upstream of GSTe2. Among these, a 2 bp adenosine indel (insertion/deletion) was found to have the greatest effect on determining promoter activity. The activity of the GSTe3 promoter was elevated to a lesser degree in the DDT-resistant strain (1.3-fold). The role of putative transcription-factor-binding sites in controlling promoter activity was investigated by sequentially deleting the promoter constructs. Several putative transcription-factor-binding sites that are responsive to oxidative stress were present within the core promoters of these GSTs, hence the effect of H2O2 exposure on the transcription of the Epsilon GSTs was investigated. In the DDT-resistant strain, expression of GSTe1, GSTe2 and GSTe3 was significantly increased by a 1-h exposure to H2O2, whereas, in the susceptible strain, only GSTe3 expression responded to this treatment.

Molecular and biochemical characterization of a sand fly population from Sri Lanka: evidence for insecticide resistance due to altered esterases and insensitive acetylcholinesterase.

With an increasing incidence of cutaneous leishmaniasis in Sri Lanka, particularly in northern provinces, insecticide-mediated vector control is under consideration. Optimizing such a strategy requires the characterization of sand fly populations in target areas with regard to species composition and extant resistance, among other parameters. Sand flies were collected by human bait and cattle-baited net traps on Delft Island, used as an illegal transit location by many refugees returning to the north of Sri Lanka from southern India where leishmaniasis is endemic. For species identification, genomic DNA was extracted and a fragment of the ribosomal 18S gene amplified. The sequence from all flies analysed matched that of Phlebotomus argentipes Annandale & Brunetti, the primary vector in India and the most likely vector in Sri Lanka. Independent morphological analysis also identified P. argentipes. To establish the current susceptibility status of vector species, data were obtained at the biochemical level, from which potential cross-resistance to alternative insecticides can be predicted. The Delft Island collection was assayed for the activities of four enzyme systems involved in insecticide resistance (acetylcholinesterase, non-specific carboxylesterases, glutathione-S-transferases and cytochrome p450 monooxygenases), establishing baselines against which subsequent collections can be evaluated. There was preliminary evidence for elevated esterases and altered acetylcholinesterase in this population, the first report of these resistance mechanisms in sand flies to our knowledge, which probably arose from the malathion-based spraying regimes of the Anti-Malarial Campaign.

Analysis of the promoters for the β-esterase genes associated with insecticide resistance in the mosquito Culex quinquefasciatus

Resistance to organophosphorus (OP) insecticides in the mosquito Culex quinquefasciatus is primarily due to the amplification and over-expression of non-specific esterases. Co-amplification of two esterase genes, estalpha2(1) and estbeta2(1), is the most common resistance genotype. In both resistant and susceptible mosquitoes the alpha- and beta-esterase genes are oriented in a head-to-head arrangement, the intergenic sequences containing promoter elements for the divergent transcription of both esterases. Transient transfection of luciferase reporter gene constructs into a C. quinquefasciatus cell line was used to study these promoters. A control vector was constructed with the strong Drosophila actin 5c promoter driving expression of beta-galactosidase. The beta-esterase promoters from both insecticide resistant and -susceptible insects were highly active in directing luciferase expression. Transfections with panels of deletions revealed several regions where binding sites for positive and negative regulatory elements are located, and candidate transcription factor sites have been identified. Gel shift assays have identified one DNA-protein interaction that is stronger with the resistant than with the equivalent but slightly altered susceptible sequence. The arthropod initiator site TCAGT 135bp upstream of the ATG in both beta-esterase promoters is essential for transcription initiation, but a putative TATA box is not involved.