Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic, metabolic and target site characterizations of three populations.

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide-treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock-down of 50% (KDT50 ) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT50 than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of ß-esterases and oxidases, but not glutathione-S-transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide-treated bednets increases.

Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypti.

Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and ß esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of ß-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.

The head and body lice of humans are genetically distinct (Insecta: Phthiraptera, Pediculidae): evidence from double infestations.

Little is known about the population genetics of the louse infestations of humans. We used microsatellite DNA to study 11 double infestations, that is, hosts infested with head lice and body lice simultaneously. We tested for population structure on a host, and for population structure among seven hosts that shared sleeping quarters. We also sought evidence of migration among louse populations. Our results showed that: (i) the head and body lice on these individual hosts were two genetically distinct populations; (ii) each host had their own populations of head and body lice that were genetically distinct to those on other hosts; and (iii) lice had migrated from head to head, and from body to body, but not between heads and bodies. Our results indicate that head and body lice are separate species.

Insecticide susceptibility in Anopheles pseudopunctipennis from Colombia: comparison between bioassays and biochemical assays.

Anopheles pseudopunctipennis, one of the primary vectors of malaria in the southwest of Colombia, was evaluated for susceptibility to the 3 major insecticide groups (organophosphates, pyrethroids, and carbamates) by bioassay and biochemical assay. Larval populations, which were collected principally from irrigation channels in agricultural areas, where the intensity of insecticide use varied, were utilized to establish susceptibility for the 1st time in this species. The baselines for each population showed a range of biological susceptibility to the insecticides evaluated, but overall no resistance was detected according to standards established by the World Health Organization. The high sensitivity of biochemical microassays enabled the detection of a small proportion of mosquitoes with higher levels of nonspecific esterases and mixed-function oxidases from 2 areas where agricultural application of organophosphate and pyrethroid insecticides had been heavy. These differences were not sufficient to affect susceptibility as measured by bioassay. No evidence of insensitive acetylcholinesterase was observed. Absence of resistance in areas that have experienced heavy insecticide application could be explained by genetic drift, by gene flow from areas without insecticide pressure, by manner of exposure to the insecticides, or by recent changes in agricultural activities that decreased insecticide use. Baseline values were established that serve as provisional susceptibility thresholds for applying simple Centers for Disease Control and Prevention biochemical assay and bioassay methods to larvae of this anopheline species.

Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets.

The permethrin tolerance (PT) of a population of the mosquito Anopheles gambiae (Diptera: Culicidae) increased following the introduction of permethrin-impregnated nets for malaria control in certain villages near Kisumu, western Kenya. Using a biochemical test that indirectly measures oxidases associated with permethrin resistance, we found that this population had higher oxidase levels than a comparison population from villages without impregnated nets. Mosquitoes from a colony of An. gambiae selected for PT, the RSP (reduced susceptibility to permethrin) strain, were exposed to permethrin with or without the oxidase inhibitor piperonyl butoxide (PB). Significantly higher mortality rates occurred when permethrin was synergized by PB, presumably by suppression of oxidases responsible for PT. An unselected (UNS) colony of An. gambiae that was more susceptible than RSP in a permethrin-susceptibility bioassay (i.e. LT50 22 min for UNS, vs. 42min for RSP) was compared with the RSP colony for levels of oxidases and esterases. The levels of both enzymes were very significantly higher in the RSP strain (P<0.0001). We speculate that use of impregnated nets selected for higher oxidase and esterase levels in An. gambiae to metabolize permethrin acquired from the nets. Both oxidase and esterase mechanisms could confer cross-resistance to other pyrethroids.

Independent selection of multiple mechanisms for pyrethroid resistance in Guatemalan Anopheles albimanus (Diptera: Culicidae).

Isofemale lines were established containing either, both, or neither of the elevated esterase and oxidase resistance mechanisms conferring pyrethroid resistance in a Guatemalan strain of Anopheles albimanus (Wiedemann). Plots of esterase and oxidase levels for individual mosquitoes from these single families correlated with data obtained using oxidase and esterase synergists in bioassays run in the bottle format. Mixed populations of pyrethroid-resistant A. albimanus adult females were selected using DDT, permethrin, or malathion; and the esterase and oxidase levels of the individual progeny were plotted. These data showed that the 3 classes of insecticide selected the 2 mechanisms differently. These results are discussed in terms of the problem of multiresistance surveillance in the field, especially concerning pyrethroid insecticides and the interaction of agricultural and public health insecticide application.

Insecticide resistance and vector control.

Insecticide resistance has been a problem in all insect groups that serve as vectors of emerging diseases. Although mechanisms by which insecticides become less effective are similar across all vector taxa, each resistance problem is potentially unique and may involve a complex pattern of resistance foci. The main defense against resistance is close surveillance of the susceptibility of vector populations. We describe the mechanisms of insecticide resistance, as well as specific instances of resistance emergence worldwide, and discuss prospects for resistance management and priorities for detection and surveillance.

Measurement of flight tone differentiates among members of the Anopheles gambiae species complex (Diptera: Culicidae).

Through digital sampling and resampling at 5,000 and 20,000 Hz of amplified mosquito flight sound, baseline separation was observed for flight tone frequency distributions of male and female Anopheles gambiae Giles, An. arabiensis Patton, An. merus Donitz, and An. melas Theobald. Males of the 4 species showed flight tones considerably higher than females. Up to 7 harmonics were measured for each species. Close correspondence for each individual mosquito of the means of the flight tone harmonics (corrected for harmonic number) demonstrated the accuracy and precision of the method. These data indicate that flight tone differences have been subjected to selection and may act as an isolating mechanism for mating or serve some other behavioral purpose in these mosquitoes. Individuals and swarms of sympatric species were distinguished from each other for both males and females, but the allopatric species, An. merus and An. melas, were indistinguishable.

Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles.

A simple method is described for treating 250-ml glass Wheaton bottles with insecticide, and using them as test chambers for detecting insecticide resistance in mosquito and sandfly populations. The methods for treating bottles, obtaining baseline data, and applying this technique to insects from the field are described. Sample data are presented from tests run on different vector species using a variety of insecticides. Time-mortality data from the bottle bioassay are presented alongside results from biochemical detection methods applied to the same mosquito population. The potential role, advantages, and limitations of the time-mortality bottle method are discussed.

Heme peroxidase activity measured in single mosquitoes identifies individuals expressing an elevated oxidase for insecticide resistance.

Optimum conditions are described for a simple, rapid, microplate-based assay that indirectly measures the differences in oxidase levels between individual susceptible, resistant, or induced mosquitoes. A small proportion (0.01-0.1) of a single mosquito is used, allowing multiple replicates of the oxidase assay. Cytochrome C is used as a positive control. The levels of oxidase found in sample populations of pyrethroid-susceptible, pyrethroid-resistant, and phenobarbital-induced Anopheles albimanus mosquitoes are characterized with the assay.

Measurement of flight tone differences between female Aedes aegypti and A. albopictus (Diptera: Culicidae).

Mosquito flight tone was amplified and digitally sampled at 20,000 samples per second (Hz). Resampling of the resulting sound files at 1,000, 5,000, and 10,000 Hz allowed comparison of flight tone frequency distributions for males and females of Aedes aegypti (L.) and A. albopictus (Skuse). Frequency distributions for females of the two species did not overlap at sampling rates of 5,000 Hz or higher, whereas considerable overlap was observed at the 1,000 Hz sampling rate. Males of the two species produced flight tones higher in frequency than those of females, but similar to each other. At the highest sampling rate, seven flight tone harmonics were measured for each species. Close correspondence of the means of the flight tone harmonics (corrected for harmonic number) demonstrated that any of the harmonics may be used accurately and precisely to calculate flight tone frequency. These data indicate that flight tone differences have been selected in these species and could act as an isolating mechanism for mating.

Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction.

A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa. The method, which is based on species-specific nucleotide sequences in the ribosomal DNA intergenic spacers, may be used to identify both species and interspecies hybrids, regardless of life stage, using either extracted DNA or fragments of a specimen. Intact portions of a mosquito as small as an egg or the segment of one leg may be placed directly into the PCR mixture for amplification and analysis. The method uses a cocktail of five 20-base oligonucleotides to identify An. gambiae, An. arabiensis, An. quadriannnulatus, and either An. melas in western Africa or An. melas in eastern and southern Africa.

A generalized approach to detection of organophosphate resistance in mosquitoes.

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.

Microplate assay of glutathione s-transferase activity for resistance detection in single-mosquito triturates.

1. Optimum conditions are described for a simple, rapid microplate assay that measures glutathione s-transferase (GST) activity accurately and precisely in small portions of single mosquito homogenates. 2. Up to 10 assay replicates were possible for individual adults and larvae. Concentration of GST activity in the head/thorax region allows blood-fed mosquitoes with abdomens removed to be used in assays. 3. The method allows the use of GST activity as a biochemical character in comparative studies of populations. 4. The microplate assay detects elevated GST activities associated with DDT resistance in Anopheles arabiensis.

Field evaluation of methods for estimating carbamate resistance in Anopheles albimanus mosquitos from a microplate assay for insensitive acetylcholinesterase.

The reliability of a published method to predict survivorship in the WHO propoxur-resistance bioassay (WHO test) from the results of a biochemical assay for detecting the insensitivity of acetylcholinesterase (AChE) is described. For biochemical assay data from three field populations of Anopheles albimanus mosquitos, the results obtained using the method did not correlate consistently with the findings of the WHO test. A modified method is then described that eliminates the effect on the assay of factors unrelated to pesticide resistance, and it is shown that this modification can be used to predict survivorship in the WHO test for mosquitos from three study sites in Guatemala. The results show that when scored visually, the insensitive AChE microplate assay is an accurate method of estimating survival in the WHO test, regardless of whether the mosquitos tested are blood-fed or not. Recommendations are given for the application and analysis of data from the insensitive AChE microplate assay for detecting and monitoring resistance to carbamate insecticides.

Biochemical resistance detection: an alternative to bioassay.

Insecticide resistance is an increasing problem in vector control programmes. Until recently, the usual means of detecting it has been by bioassay, requiring the use of relatively large numbers of insects and insecticide-impregnated test papers which may be difficult to prepare and store reproducibly. William Brogdon argues for the use of biochemical microplate assays which are cheaper and easier to use, permit up to 30 assays to be made on a single insect, and give more reproducible results.

Effect of temperature on an enzyme assay to detect fenitrothion resistance in Anopheles albimanus mosquitos.

A laboratory strain of Anopheles albimanus Wiedemann of known fenitrothion resistance was used in the field to compare the results of the WHO test for determining fenitrothion resistance in mosquitos with those of an enzyme microplate assay. The level of resistance obtained with the enzyme assay increased with the ambient temperature, and in order to compensate for this temperature effect, the incubation time was reduced. With the adjusted incubation times, the results for the microassay from 23 degrees C to 32 degrees C were the same as those found with the WHO test. The fenitrothion resistance of a field population of A. albimanus mosquitos determined between 27 degrees C and 31 degrees C using the adjusted enzyme microassay or the WHO test did not differ in a statistically significant way.