Biomedical subjects
Publications and source records attributed to J Hemingway.
Treatment of head louse infection is primarily through topical insecticides. However, there is growing evidence of resistance. A representative population sample was tested using biochemical and molecular methods; it was shown that, in Wales, treatments containing pyrethroids are likely to be less effective in controlling head louse infection than those containing organophosphates.
We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae, important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides.
Malaria control in southern Mozambique is currently by indoor residual carbamate insecticide treatment, with pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles funestus s.s. to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 19 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. funestus from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A high level of pyrethroid resistance was detected in An. funestus populations in southern Mozambique. An. funestus outside Maputo province were still susceptible to pyrethroids. An. funestus from six localities also were resistant to carbamate insecticides propoxur and bendiocarb. Both pyrethroid and carbamate resistance occurred in five of these six localities. Mosquitoes from five of the localities with elevated p450 estimates, compared with the insecticide-susceptible Durban strain, were pyrethroid-resistant. The only exception to this trend was Mozal, which had elevated p450 estimates but full pyrethroid susceptibility by bioassay. The lack of cross-resistance between pyrethroids and DDT in Mozambican An. funestus suggests that a kdr-type target site resistance mechanism has not been selected. Low levels of insecticide-insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in all populations tested. The high level of metabolically based pyrethroid resistance has implications for current malaria control programs in Mozambique.
Malaria control in the southern part of Mozambique is currently by indoor residual spraying with a carbamate insecticide and by pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles gambiae s.s. and Anopheles arabiensis Patton to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 17 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. gambiae s.s. from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A low level of pyrethroid resistance was detected in populations in southern Mozambique. Populations outside Maputo province were still susceptible to pyrethroids. Low level resistance to the carbamate propoxur also was detected in An. arabiensis from two localities. Mosquitoes from five of the localities had elevated p450 estimates, compared with the insecticide susceptible Durban strain. The lack of cross-resistance between pyrethroids and DDT in Mozambican populations suggests that a kdr-type target site resistance mechanism has not been selected. Increased frequencies of insecticide insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in 16 of the populations tested. Although vector control with bendiocarb is not being compromised by the presence of the acetylcholinesterase mechanism alone, the high level of insensitive acetylcholinesterase unless sensibly managed may have long-term implications for malaria control programs in Mozambique.
The malaria situation in Sri Lanka worsened during the 1990s with the emergence and spread of resistance to the drugs and insecticides used for control. Chloroquine resistance has increased rapidly over this period, but adverse changes in malaria transmission are more closely associated with insecticide use rather than drug resistance. Insecticide susceptibility tests were routinely carried out in key anopheline vectors across the country for more than a decade. These sentinel data were combined with data collected by other research programmes and used to map the spatial and temporal trends of insecticide resistance in the main vectors, Anopheles culicifacies and A. subpictus, and to examine the relationship between insecticide resistance, changes in national spraying regimens and malaria prevalence. Both species had widespread resistance to malathion, the insecticide of choice in the early 1990s. Both species were initially susceptible to the organophosphate and pyrethroid insecticides used operationally from 1993, but some resistance has now been selected. The levels of malathion and fenitrothion resistance in A. subpictus were higher in some ecological regions than others, which may be related to the distribution of sibling species, agricultural pesticide exposure and/or environmental factors. The study highlights that the emergence and spread of insecticide resistance is a constant threat and that active surveillance systems are vital in identifying key vectors and evidence of resistance.
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.
Glutathione transferases (GSTs) are a diverse family of enzymes found ubiquitously in aerobic organisms. They play a central role in the detoxification of both endogenous and xenobiotic compounds and are also involved in intracellular transport, biosynthesis of hormones and protection against oxidative stress. Interest in insect GSTs has primarily focused on their role in insecticide resistance. GSTs can metabolize insecticides by facilitating their reductive dehydrochlorination or by conjugation reactions with reduced glutathione, to produce water-soluble metabolites that are more readily excreted. In addition, they contribute to the removal of toxic oxygen free radical species produced through the action of pesticides. Annotation of the Anopheles gambiae and Drosophila melanogaster genomes has revealed the full extent of this enzyme family in insects. This mini review describes the insect GST enzyme family, focusing specifically on their role in conferring insecticide resistance.
A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families.
AnoBase (http://www.anobase.org) is an integrated, relational database of basic biological and genetic data on anopheline species, with a particular emphasis on Anopheles gambiae. It has been designed as an information source and research support tool for the broad vector biology community. Although AnoBase is not a primary genomic database that develops and provides tools to access the genome of the malaria mosquito, it nevertheless contains several sections that offer data of genomic interest such as in situ hybridization images, an integrated gene tool and direct online access to AnoXcel, the proteomic database of An. gambiae. Moreover, AnoBase also contains information on non-gambiae mosquito species and a novel section on studies related to insecticide resistance.
Insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides have become one of the most promising interventions to prevent malaria in highly endemic areas. Despite the large body of experience documenting their health impact and the best way to distribute them, some key practical issues remain unresolved. For example, the duration of effective life of a net under field conditions is unknown. The most important factor affecting net effectiveness is the issue of regular re-treatment with insecticide. Washing is also an important determinant of insecticide longevity in the field. Trials were undertaken to provide some essential field information on ITNs within the site of an extended ITN programme in the Morogoro region of Tanzania. It was found that 45% of all nets were in bad condition (defined as more than seven large holes). It is concluded that an effective 'life' for polyester nets is 2-3 years. Further, two-thirds of the 20% of nets that were reported as having been re-treated within the last 12 months had less than 5 mg/m(2) of insecticide. According to the World Health Organization this is insufficient to be effective. People reported that they washed their nets four to seven times per year, usually with soap. Observations showed that such washing does not harm the nets and that the wash-water was unlikely to have an impact on the environment. Finally, bioassays were carried out with Anopheles gambiae on polyester netting with 0.5, 2, 5, 10 and 30 mg/m(2) of deltamethrin, alphacypermethrin and lambdacyhalothrin to assess the effectiveness of pyrethroids. The results confirmed that even with low insecticide concentrations, nets can still provide partial protection.
Resistance to permethrin in an East African population of the major malaria vector, Anopheles gambiae is multifactorial. A mutated sodium channel allele and enhanced insecticide metabolism contribute to the resistance phenotype. We used microsatellite markers to scan the genome for quantitative trait loci (QTL) associated with permethrin resistance. Two major and one minor QTL were identified. The first QTL, rtp1, colocalizes with the sodium channel gene on chromosome 2L thus further supporting the importance of mutations in this gene in conferring permethrin resistance. The second two loci are located on the third chromosome and one of these, rtp2, flanks a large cluster of cytochrome P450 genes. Further detailed mapping of these regions will help elucidate the molecular mechanisms of metabolic resistance to insecticides.
During extensive sampling in Burkina Faso and other African countries, the Leu-Phe mutation producing the kdr pyrethroid resistance phenotype was reported in both Anopheles gambiae ss and A. arabiensis. This mutation was widely distributed at high frequency in the molecular S form of A. gambiae while it has been observed at a very low frequency in both the molecular M form and A. arabiensis in Burkina Faso. While the mutation in the M form is inherited through an introgression from the S form, its occurrence is a new and independent mutation event in A. arabiensis. Three nucleotides in the upstream intron of the kdr mutation differentiated A. arabiensis from A. gambiae ss and these specific nucleotides were associated with kdr mutation in A. arabiensis. Ecological divergences which facilitated the spread of the kdr mutation within the complex of A. gambiae ss in West Africa, are discussed.
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.
The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so-called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr-like pyrethroid-resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid-selected strain from Dubai (DUB-R) were 182-fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid-resistance (i.e. esterases, monooxygenases and glutathione S-transferases) were significantly higher in the permethrin-resistant than in the susceptible strain, but the use of synergists--piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases--did not fully prevent resistance in larvae (permethrin LC50 reduced by only 51-68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237-bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage-gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A-T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid-resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr-like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.
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In Culex mosquitoes (Diptera: Culicidae), the most common mechanism for resistance to organophosphorus (OP) insecticides involves amplification of one or more esterases. Two esterase loci are often involved, with different allelic forms co-amplified. Estalpha3(1) is co-amplified with estbeta1 in a Colombian (COL) strain of Culex quinquefasciatus Say. These two alleles co-migrate on acrylamide gels, often leading to misscoring of the phenotype as elevation of a single estbeta enzyme. By sequencing COL genomic DNA, we determined the estalpha3(1) gene length is 1623 nucleotides. The open reading frame of estalpha3(1) encodes a 540 amino acid protein, as for estalpha2(1) in strain Pel RR from Sri Lanka. The intron/exon boundaries of estalpha3(1) are identical to those of estalpha2(1), suggesting that they are alleles of the same locus. The COL estalpha3(1) gene differs from estaalpha3(2) in strain MRES from Cuba, although they have equivalent electrophoretic mobility, showing that these two strains contain distinct resistance-associated amplicons. Twenty nucleotide differences were scored between the MRES partial 495 bp sequence and that in the COL strain, with two amino acid changes, demonstrating distinct estalpha enzymes. Our sequencing data show 95% identity between the three estalpha genes (each has six introns and seven exons) in OP-resistant Cx. quinquefasciatus. Amplified estalpha3(1) and estbeta1 are at least 10kb apart in temephos-selected COL and 2.7kb apart in Pel RR, whereas these non-amplified genes are only 1.7kb apart in the nonselected parental COL stock, as in Pel SS (susceptible Sri Lankan strain), demonstrating that this region of the genome is susceptible to expansion and contraction.
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.
Cytochrome P450s are a superfamily of haemoproteins, important in the metabolism of endogenous compounds and xenobiotics. As a first step to elucidating the role of this family in insecticide resistance in the malaria mosquito, Anopheles gambiae, we have cloned and mapped multiple P450 genes. Sixteen cDNAs encoding full-length P450s were cloned and physically mapped to the mosquito's polytene chromosomes. Fourteen of these encode putative CYP6 proteins and two encode P450s belonging to the CYP9 class. Eighteen new A. gambiae Cyp4 P450 genes were identified using degenerate PCR primers, cDNAs were detected for ten and in situ locations for thirteen members of this gene family.