Martin S. Williamson

Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s. s.

Pyrethroid‐impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage‐gated sodium channel protein recently shown to be associated with mutations of the para‐type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR‐based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.

Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides.

We report the isolation of cDNA clones containing the full 6.3-kb coding sequence of thepara-type sodium channel gene of the housefly,Musca domestica. This gene has been implicated as the site of knockdown resistance (kdr), an important resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. The cDNAs predict a polypeptide of 2108 amino acids with close sequence homology (92% identity) to theDrosophila para sodium channel, and around 50% homology to vertebrate sodium channels. Only one major splice form of the housefly sodium channel was detected, in contrast to theDrosophila para transcript which has been reported to undergo extensive alternative splicing. Comparative sequence analysis of housefly strains carryingkdr or the more potentsuper-kdr factor revealed two amino acid mutations that correlate with these resistance phenotypes. Both mutations are located in domain II of the sodium channel. A leucine to phenylalanine replacement in the hydrophobic IIS6 transmembrane segment was found in two independentkdr strains and sixsuper-kdr strains of diverse geographic origin, while an additional methionine to threonine replacement within the intracellular IIS4-S5 loop was found only in thesuper-kdr strains. Neither mutation was present in five pyrethroid-sensitive strains. The mutations suggest a binding site for pyrethroids at the intracellular mouth of the channel pore in a region known to be important for channel inactivation.

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica).

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.

A sodium channel point mutation is associated with resistance to DDT and pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

The voltage‐gated sodium channel is the primary target site of DDT and pyrethroid insecticides, and point mutations in the domain II region of the channel protein have been implicated in the knockdown resistant (kdru200a) phenotype of several insect species. Here, we report that one of these mutations, a leucine‐to‐phenylalanine replacement in transmembrane segment IIS6, is also found in certain insecticide‐resistant clones of the peach‐potato aphid, Myzus persicae. The mutation was present in four clones with amplified E4 esterase genes, but was absent from both susceptible clones and those with amplified FE4 genes. The inferred presence of kdr‐type resistance in the four E4 clones was subsequently confirmed by bioassays that showed this to be the primary mechanism of resistance to deltamethrin and DDT, although the esterase‐based mechanism also contributes to the overall level of deltamethrin resistance. The kdr mutation on its own conferred 35‐fold resistance to deltamethrin and this was enhanced up to 540‐fold when it was present in a high (E4) esterase background. The esterase (FE4) mechanism was far less effective without the kdr mutation, conferring just 3–4‐fold resistance to deltamethrin. These findings, and the linkage disequilibrium of the kdr mutation within clones overproducing the E4 esterase, have important implications for the evolution of resistance in this insect and for the use of pyrethroid sprays in the management of M. persicae populations in the field.

Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes.

We have identified cDNA clones coding for the major sulphur‐rich and sulphur‐poor groups of barley storage proteins (the B‐ and C‐hordeins, respectively). Hybridization studies have revealed unexpected homologies between B‐ and C‐hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C‐hordein‐related sequences within, or closely associated with, B‐hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B‐hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline‐rich, sulphur‐poor), which is homologous to C‐hordein sequences, and domain 2 (non‐repetitive, proline‐poor, sulphur‐rich), which makes up two‐thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B‐ and C‐hordein mRNAs have an asymmetric base composition (>80% C‐A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro‐Gln‐Gln‐Pro‐Phe‐Pro‐Gln‐Gln. We discuss the evolutionary implications of the domain structure of the B‐hordeins and the unusual relationship between the two groups of barley storage proteins.

A comparative study of voltage-gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species

We report the complete cDNA sequence of the Anopheles gambiae voltage‐gated sodium channel (VGSC) α‐subunit isolated from mature adult mosquitoes. The genomic DNA contains 35 deduced exons with a predicted translation of ≤ 2139 amino acid cDNAs. The transcription of the gene is, however, complex, alternate splicing being evident for at least five optional exons (or exon segments) and two sets of mutually exclusive exons. Overall gene organization was also compared with that of other VGSCs within the Insecta. Several insecticides used in mosquito control (including DDT and synthetic pyrethroids) target the VGSC. Isolation of the sodium channel cDNA for An. gambiae: (1) allows prediction of likely single nucleotide polymorphisms that may arise at residue L1014 to cause resistance to insecticides; (2) defines An. gambiae exon usage in key areas of the VGSC protein that are known (from previous studies in a range of different pest species) to have roles in altering insecticide susceptibility and in generating resistance; and (3) is a critical first step towards development of refined malarial control strategies and of new diagnostics for resistance monitoring.

Analogous pleiotropic effects of insecticide resistance genotypes in peach–potato aphids and houseflies

We show that single-point mutations conferring target-site resistance (kdr) to pyrethroids and DDT in aphids and houseflies, and gene amplification conferring metabolic resistance (carboxylesterase) to organophosphates and carbamates in aphids, can have deleterious pleiotropic effects on fitness. Behavioural studies on peach–potato aphids showed that a reduced response to alarm pheromone was associated with both gene amplification and the kdr target-site mutation. In this species, gene amplification was also associated with a decreased propensity to move from senescing leaves to fresh leaves at low temperature. Housefly genotypes possessing the identical kdr mutation were also shown to exhibit behavioural differences in comparison with susceptible insects. In this species, resistant individuals showed no positional preference along a temperature gradient while susceptible genotypes exhibited a strong preference for warmer temperatures.

Single blind, randomised, comparative study of the Bug Buster kit and over the counter pediculicide treatments against head lice in the United Kingdom

Abstract Objective To compare the effectiveness of the Bug Buster kit with a single treatment of over the counter pediculicides for eliminating head lice. Design Single blind, multicentre, randomised, comparative clinical study. Setting Four counties in England and one county in Scotland. Participants 133 young people aged 2-15 years with head louse infestation: 56 were allocated to the Bug Buster kit and 70 to pediculicide treatment. Interventions Home use of proprietary pediculicides (organophosphate or pyrethroid) or the Bug Buster kit. Main outcome measure Presence of head lice 2-4 days after end of treatment: day 5 for the pediculicides and day 15 for the Bug Buster kit. Results The cure rate using the Bug Buster kit was significantly greater than that for the pediculicides (57% v 13%; relative risk 4.4, 95% confidence interval 2.3 to 8.5). Number needed to treat for the Bug Buster kit compared with the pediculicides was 2.26. Conclusion The Bug Buster kit was the most effective over the counter treatment for head louse infestation in the community when compared with pediculicides.

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects†

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained susceptible Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed.

Reduced alarm response by peach–potato aphids, Myzus persicae (Hemiptera: Aphididae), with knock-down resistance to insecticides ( kdr )may impose a fitness cost through increased vulnerability to natural enemies

Termites were sampled using randomized soil pits in 64 cropping plots, each 25 x 25 m, forming an experimental agrisilvicultural system in both a 6- and an 18-year-old Terminalia ivorensis plantation, in which canopy cover, crop, cropping system and land preparation were the principal treatment variables. The treatments were established in April 1995 and sampling was carried out in November 1995, February 1996 and July 1996. A total of 82 termite species were found, of which 67 were soil-feeders. Overall termite abundance and the abundance of soil-feeders increased between November 1995 and July 1996, reaching a mean of nearly 6000 m -2 . Pooling termite data from these sampling dates, in the old plantation, the high canopy cover treatment (192 stems ha -1 ) had a greater abundance of termites, compared with the low canopy cover treatment (64 stems ha -1 ) and this effect was independent of crop type (plantain or cocoyam), cropping system (single stands or mixed crops) and land preparation (mulch retained or burned, plantain only). The young tree plantation (same tree densities as in the old plantation) showed no significant difference in termite abundance between high and low canopy (levels of tree foliage) densities, though the high canopy sheltered a greater number of termites. Analysis of covariance showed that crop yield (both plantain and cocoyam) was not directly linked to the abundance of all termite populations, but that the cocoyam yield was positively correlated with the abundance of soil-feeding termites (the majority in the assemblage) in the young plantation. This may be due to the beneficial conditioning of soil resulting from the foraging and construction activities of soil-feeders.