Detection of Pyrethroid Resistance Mutations in the Major Leishmaniasis Vector Phlebotomus papatasi.

Phlebotomine sand flies (Diptera: Psychodidae) are primary vectors of leishmaniasis. Greece and Turkey are both endemic for visceral and cutaneous leishmaniasis and are widely affected by the disease. Measures commonly applied for controlling sand flies rely on the use of insecticides, predominantly pyrethroids. A worldwide problem associated with the intensive use of insecticides is the development of resistance. Scarce information is available regarding the resistance status in sand fly populations. Sand flies were collected from Greece (Thessaloniki, Peloponnese, Chios island) and Turkey (Sanliurfa) and analyzed for the presence and frequency of target-site knockdown resistance mutations on the voltage-gated sodium channel (Vgsc) gene. Five sand fly species were included in the analysis: Phlebotomus perfiliewi Parrot, Phlebotomus neglectus Tonnoir, Phlebotomus simici Nitzulescu, Phlebotomus tobbi Adler and Theodor, and Phlebotomus papatasi Scopoli. Their Vgsc gene-domain II was analyzed for the presence of known pyrethroid resistance mutations. The mutation 1014F, associated with pyrethroid-resistant phenotypes, was detected in P. papatasi sand flies from Sanliurfa at an allele frequency of 48%. Homozygotes for the wild type allele 1014L (Leu/Leu) represented 36% of the population, while homozygotes for the resistant allele 1014F (Phe/Phe) and heterozygotes encompassing both alleles (Leu/Phe) each had a frequency of 32%. In all other sand fly species, only the wild type allele 1014L was detected. This is the first report for the detection of resistance mutations in the major leishmaniasis vector P. papatasi and is of major concern regarding leishmaniasis control.

Effect of DMI-resistance mechanisms on cross-resistance patterns, fitness parameters and aflatoxin production in Aspergillus parasiticus Speare.

Aspergillus parasiticus mutant strains resistant to DMIs were isolated in a high mutation frequency after UV-mutagenesis and selection on media containing flusilazole. Two different resistant phenotypes, R(1) and R(2), on the basis of their aflatoxigenic ability were identified. All R(1) mutant strains produced aflatoxins at concentrations significantly higher (up to 3-fold) than the wild-type parent strain on yeast extract sucrose medium, whereas the majority of mutant strains (R(2) phenotype) lost their aflatoxigenic ability. Real-time PCR analysis of the expression levels of the aflR gene, a pathway transcriptional regulatory gene in aflatoxin biosynthesis, showed that this gene was not expressed in R(2) mutant strains tested. Study of fitness determining parameters showed that most flusilazole-resistant mutant strains had mycelial growth rate, sporulation and spore germination lower that the sensitive one. Cross-resistance studies with other fungicides showed that all R(1) mutant strains were also resistant to the DMIs imazalil and tebuconazole, but retained their parental sensitivity to fungicides affecting other metabolic pathways and/or cellular processes. Contrary to the above, all R(2) mutant strains exhibited a low to moderate multi-drug resistance to DMIs and to several other fungicide classes. Two different homologous genes, cyp51A and cyp51B, encoding C-14 alpha sterol demethylase (Cyp51) and an mdr gene encoding an ATP-binding cassette protein which may be involved in multidrug resistance were cloned and characterized. Sequence comparison of cyp51A gene revealed an amino acid substitution from glycine (GGG) to tryptophan (TGG) at position 54 (G54W) in two out of three of R(1) mutant strains. Analysis of deduced amino acid sequence of cyp51B showed that no mutations were associated with DMI resistance. Study for the transcriptional levels of cyp51A showed that this gene was over-expressed in the third aflatoxigenic mutant strain. Neither amino acid substitutions nor an overexpression of the cyp51A gene were found in the R(2) mutant strains tested. Real-time PCR analysis showed high levels (up to 25-fold higher) of the mdr transcript in all R(2) mutant strains tested. This is the first report describing the existence of two cyp51 genes and a potential mdr gene coding for an ATP binding cassette protein in A. parasiticus. These results also indicate that multiple biochemical mechanisms, including target-site modification due to mutation at cyp51A gene, overexpression of cyp51A gene and the function of an ABC transporter protein, are responsible for DMI-resistance in A. parasiticus. Our findings suggest that A. parasiticus have the genetic and biochemical potential for the appearance of highly aflatoxigenic DMI-resistant isolates in the field.

Geographical distribution and evolutionary history of organophosphate-resistant Ace alleles in the olive fly (Bactrocera oleae).

Acetylcholinesterase (Ace) is the molecular target of organophosphate (OP) insecticides, and two mutations that confer different levels of OP insensitivity have previously been identified in the olive fly, Bactrocera oleae. Numerous sensitive and two insensitive alleles (including one convergent acquisition) are described from the entire worldwide distribution of the fly. Most of the variation is harbored in the native range of the species and in the Middle East and consists of numerous low-frequency sensitive alleles. The insensitive alleles likely came to high frequency more recently in the Mediterranean region or in the Middle East, reaching frequencies as high as 100% in some populations, and determined a corresponding decline in overall genetic variation. We hypothesize that the major force that shaped the current distribution of resistant and non-resistant acetylcholinesterase alleles is natural selection, likely responsible for the high frequency of insensitive alleles in areas where organophosphates have been used extensively. We also discuss a role for historical contingency, that can explain why sensitive alleles are absent altogether in the species ancestral range and present in areas of recent expansion, such as California, despite the limited use of OPs.

Molecular characterization of the amplified aldehyde oxidase from insecticide resistant Culex quinquefasciatus.

Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.