S. H. P. P. Karunaratne

Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estalpha2(1) and estbeta2(1) genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estalpha2(1) and estbeta2(1) are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estbeta2(1) gene from this amplicon has greater transcription than estalpha2(1) in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.

Insecticides and mosquito-borne disease.

The primary means of controlling mosquito-borne diseases such as malaria and filariasis is still by residual spraying with insecticides. Here we show that insecticide-resistant Culex quinquefasciatus mosquitoes are less likely to transmit filariasis than their insecticide-susceptible counterparts. If this surprising finding extends to other combinations of insect species, insecticide-resistance mechanisms and disease, it could have widespread consequences for the control of vector-borne disease.

Malathion resistance and prevalence of the malathion carboxylesterase mechanism in populations of mosquito vectors of disease in Sri Lanka

OBJECTIVE To determine the levels of malathion resistance and prevalence of the malathion carboxylesterase mechanism among mosquitoes in Sri Lanka. METHODS Bioassays were carried out using WHO-recommended methods on samples of the following Sri Lankan mosquito vectors: Culex quinquefasciatus, C. tritaeniorhynchus, C gelidus, Anopheles culicifacies B, A. subpictus, Aedes aegypti and A. albopictus. FINDINGS Malathion-specific carboxylesterase mechanisms were found in A. culicifaies and A. subpictus, both giving high rates of insecticide metabolism. In contrast, malathion resistance in C. quinquefasciatus and C. tritaeniorhynchus is linked to broad-spectrum resistance to organophosphorus compounds due to elevated levels of esterases that sequester malaoxon, but are unable to metabolize malathion. CONCLUSIONS Resistance among the Anophelesspp. must have occurred as a direct result of antimalarial activities, since malathion use in Sri Lanka is limited to public health treatments. In contrast, resistance among Culex spp. has resulted from large-scale use of the organophosphorus insecticide group as larvicides for filariasis control and on rice paddy, where C tritaeniorhynchus predominantly breeds, for agricultural purposes.

Multiple insecticide resistance mechanisms involving metabolic changes and insensitive target sites selected in anopheline vectors of malaria in Sri Lanka

BackgroundThe current status of insecticide resistance and the underlying resistance mechanisms were studied in the major vector of malaria, Anopheles culicifacies, and the secondary vector, Anopheles subpictus in five districts (Anuradhapura, Kurunegala, Moneragala, Puttalam and Trincomalee) of Sri Lanka. Eight other anophelines, Anopheles annularis, Anopheles barbirostris, Anopheles jamesii, Anopheles nigerrimus, Anopheles peditaeniatus, Anopheles tessellatus, Anopheles vagus and Anopheles varuna from Anuradhapura district were also tested.MethodsAdult females were exposed to the WHO discriminating dosages of DDT, malathion, fenitrothion, propoxur, λ-cyhalothrin, cyfluthrin, cypermethrin, deltamethrin, permethrin and etofenprox. The presence of metabolic resistance by esterase, glutathione S-transferase (GST) and monooxygenase-based mechanisms, and the sensitivity of the acetylcholinesterase target site were assessed using synergists, and biochemical, and metabolic techniques.ResultsAll the anopheline species had high DDT resistance. All An. culicifacies and An. subpictus populations were resistant to malathion, except An. culicifacies from Kurunegala, where there was no malathion carboxylesterase activity. Kurunegala and Puttalam populations of An. culicifacies were susceptible to fenitrothion. All the An. culicifacies populations were susceptible to carbamates. Both species were susceptible to the discriminating dosages of cypermethrin and cyfluthrin, but had different levels of resistance to other pyrethroids. Of the 8 other anophelines, only An. nigerrimus and An. peditaeniatus were resistant to all the insecticides tested, probably due to their high exposure to the insecticides used in agriculture. An. vagus showed some resistance to permethrin.Esterases, GSTs and monooxygenases were elevated in both An. culicifacies and An. subpictus. AChE was most sensitive to insecticides in Kurunegala and Trincomalee An. culicifacies populations and highly insensitive in the Trincomalee An. subpictus population.ConclusionThe complexity of the resistance segregating in these field populations underlines the need for new molecular tools to identify the genomic diversity, differential upregulation and different binding specificities of resistance conferring genes, and the presence of different subspecies with different vectorial capacities.

Japanese encephalitis in Sri Lanka: comparison of vector and virus ecology in different agro-climatic areas

The ecology of Japanese encephalitis (JE) in different agro-climatological areas of Sri Lanka was studied in relation to the abundance of mosquito vectors, infection in domestic livestock, and human infection and disease. There was an inverse correlation between altitude and the abundance of potential JE vectors, as well as JE seroprevalence in domestic livestock and in man. Little or no JE infection was documented above 1200 m elevation. JE seroprevalences in cattle and goats were better predictors of human infection risk than was porcine seroprevalence. In areas with asynchronous porcine infection occurring over many months, high overall JE seroprevalence in pigs was found with little evidence of human infection. Porcine JE infection occurring in synchronous bursts associated with monsoonalrains was correlated with significant bovine, ovine and human seroprevalence in 2 low elevation study areas, Anuradhapura (dry zone) and Ragama (wet zone), with epidemic human JE in the former area and endemic disease in the latter.

Insecticide resistance spectra and resistance mechanisms in populations of Japanese encephalitis vector mosquitoes, Culex tritaeniorhynchus and Cx. gelidus, in Sri Lanka

Culex tritaeniorhynchus Giles and Cx. gelidus Theobald (Diptera: Culicidae), both vectors of Japanese encephalitis, were collected in 1984 and 1998 from two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Using wild‐caught adult mosquitoes from light traps, log dosage‐probit mortality curves for insecticide bioassays were obtained for three insecticides: malathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was ∼100‐fold more resistant to malathion and 10‐fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynchus breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in other types of aquatic habitats less prone to pesticide applications. Resistance in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. gelidus remained predominantly susceptible.

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.

Amplification of a serine esterase gene is involved in insecticide resistance in Sri Lankan Culex tritaeniorhynchus

Culex tritaeniorhynchus, the major vector of Japanese encephalitis in Sri Lanka, is resistant to organophosphorus insecticides, with a 10‐fold resistance ratio at the LC50 for chlorpyrifos, and a high heterogeneity factor in the insect field population. The major mechanism of resistance in this species, as in the mosquito C. quinquefasciatus, is elevation of esterase activity. Basic biochemical, immunological and molecular analysis suggests that the C. tritaeniorhynchus CtrEstβ1 gene is orthologous to the C. quinquefasciatus amplified Est7beta;s. The Estβ21 antiserum cross‐reacts strongly with CtrEstβ11. Its corresponding cDNA, over the 545 base pairs sequenced, has ∼84% identity with the various C. quinquefasciatus Estβs. The gene is amplified in C. tritaeniorhynchus. Amplification of the same esterase in two independent species, along with multiple amplification events involving this esterase gene in C. quinquefasciatus suggests that the location of this gene within the genome predisposes it to amplification.

Determination of the role of elevated B2 esterase in insecticide resistance in Culex quinquefasciatus (Diptera: Culicidae) from studies on the purified enzyme

A method for the purification of elevated esterase B 2 from the Sri Lankan Pel RR strain of Culex quinquefasciatus (Say) has been developed, using sequential column chromatography. The pure enzyme is stable, with no decrease in specific activity after several months, if stored in the presence of 25 mM dithiothreitol and 50% glycerol at –20°C. The enzyme has a pl of 5 and an estimated molecular weight for the monomeric enzyme of 63 kD from SDS-PAGE. In all Culex strains with elevated B 2 , another esterase (A 2 ) is always co-elevated. The strains with these esterases are resistant to a broad range of organophosphorus insecticides. The previous purification and characterization of A 2 from Pel RR indicated that the role of this enzyme was primarily to sequester rather than metabolize organophosphates. Purified B 2 , rapidly binds malaoxon, the activated form of malathion, but not malathion itself. The turnover rate of bound malaoxon is slow, suggesting that the role of this esterase in malathion resistance is also sequestration. The similar bimolecular rate constants for both A 2 and B 2 with malaoxon suggest both esterases are important in resistance, which may explain the almost complete linkage disequilibrium that the elevated forms of these two esterases are maintained in when under pesticide selection pressure.

Characterization of the elevated esterase-associated insecticide resistance mechanism in Nilaparvata lugens (Stal) and other planthopper species

Insecticide-resistant strains of the brown planthopper, Nilaparvata lugens, from four locations all had a single diffuse elevated esterase band on native polyacrylamide gel electrophoresis. The white backed planthopper Sogatella furcifera had two elevated esterases with lower relative mobilities than the N. lugens esterase, while the insecticide-susceptible N. bakeri and the grass-associated sibling species of N. lugens sensu lato all had a single low intensity staining esterase with a lower relative mobility than the resistance associated N. lugens esterase. All the esterases were inhibited by pre-incubation with 0.1 mM paraoxon, but were not affected by permethrin up to its solubility limit, indicating their possible role in organophosphorus, but not pyrethroid insecticide, resistance. Partial purification of the elevated esterase from insecticide resistant Sri Lankan N. lugens showed that despite its diffuse nature on gels it purified as a single isoform, with a specific activity of 5.85 mumol min- 1 m...