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.

Long-term use of permethrin-impregnated nets does not increase Anopheles gambiae permethrin tolerance.

Previous use of permethrin-impregnated bednets (mosquito nets) and curtains in four Kenyan villages for one year, 1990-91, raised the permethrin LT50 of Anopheles gambiae to 2.4-fold above its baseline value, designated permethrin tolerance (PT), as measured by exposure to 0.25% permethrin-impregnated papers in W.H.O. test-kits. During 1992-93, with ongoing use of permethrin-impregnated nets and curtains, PT regressed slightly compared with the contemporary susceptibility level of An.gambiae from non-intervention villages, to 1.8-fold in 1992 and only 1.6-fold in 1993. Thus the selection pressure of impregnated nets for PT in An.gambiae appears to be minimal in our study villages, although the impact of permethrin was demonstrated by a significantly lower parous-rate of An.gambiae females in the intervention (63-66%) than in non-intervention (79%) villages, and by reduced malaria transmission (reported elsewhere). In a selected stock of An.gambiae from the study area, PT did not affect the susceptibility to deltamethrin, fenitrothion, propoxur or DDT. Bioassays described herein provide easy procedures for field-monitoring of mosquito susceptibility/tolerance/resistance to insecticides used for net impregnation in operational programmes.

Effects of permethrin-impregnated bed nets on malaria vectors of northern Guatemala.

The authors evaluated the effects on malaria vectors of bed nets impregnated with permethrin over the course of a 16-month controlled study in four communities of Northern Guatemala. Anopheles albimanus and An. vestitipennis were the known malaria vectors in the area. Households were allocated to one of three experimental groups: those receiving bed nets impregnated with 500 mg/m2 of permethrin, those receiving untreated bed nets, and those where no intervention measures were taken. The impact of the treated and untreated bed nets on mosquito abundance, behavior, and mortality was determined by indoor/outdoor night-bite mosquito collections, morning pyrethrum spray collections, inspection of bed net surfaces for dead mosquitoes, and capture-release-recapture studies. The duration of the treated nets' residual insecticide effect was assessed by modified WHO cone field bioassays, and their pyrethrin content was estimated by gas-liquid chromatography analysis. The most important observation was that fewer mosquitoes were found to be resting in the households with treated bed nets. The treated nets probably functioned by both repelling and killing vector mosquitoes. Capture-release-recapture studies showed exit rates from houses with treated nets were higher (94%) than those from control houses (72%), a finding that suggests repellency. However, no significant differences were noted between the indoor night-bite mosquito collections at houses with and without treated nets. The horizontal surfaces of treated bed nets were nearly 20 times more likely to contain dead anopheline mosquitoes than were the comparable surfaces of untreated nets. the bioassays indicated that unwashed permethrin-impregnated bed nets retained their insecticidal activity for 6 months after treatment.

Reduced susceptibility of Anopheles gambiae to permethrin associated with the use of permethrin-impregnated bednets and curtains in Kenya.

Susceptibility of the malaria vector Anopheles gambiae to permethrin decreased following the installation of mosquito nets impregnated with 0.5 g permethrin per square metre in four villages near Kisumu, Kenya. During the first year that permethrin-impregnated bednets and curtains were in place, the exposure time to 50% mortality (LT50) increased 2.5-fold from 13 to 33 min, while the LT50 for An.gambiae was unchanged in two other villages where no intervention measures were used. Two years after permethrin-impregnated mosquito nets were distributed the LT50s for An.gambiae were 28, 28 and 16 min, respectively, in the villages with bednets, curtains and with no such intervention. Using a colony of An.gambiae derived from females collected in the villages using permethrin-impregnated mosquito nets, we lengthened the LT50 from 28 to 41 min in two generations by exposing all females to permethrin-treated papers for 60 min and rearing offspring of the survivors. Permethrin-impregnated bednets and curtains are intended to reduce vectorial capacity. Reduced susceptibility to permethrin could counter this beneficial effect.

Effectiveness of permethrin-impregnated bed nets and curtains for malaria control in a holoendemic area of western Kenya.

The effectiveness of village-wide use of permethrin-impregnated bed nets or eave, window, and door curtains as control measures for Plasmodium falciparum malaria was evaluated during two successive high-transmission seasons in western Kenya. Pairs of villages were assigned to one of three study groups: bed net, curtain, or control. Clinical, parasitologic, and entomologic measures were made from March to July 1990 and again 12 months later. When compared with the controls in 1990 and 1991, we observed a marked reduction in the incidence of P. falciparum infections in children less than six years old in the bed net villages (reduced by 40% and 48%) and a smaller but still significant reduction in the curtain villages (10% and 33%). Significant reductions were also seen in the incidence of P. falciparum parasitemias greater than 2,500/mm3 in the bed net group (reduced by 44% and 49%) and curtain group (16% and 32%). Additionally, we observed significant reductions in the incidence of documented fevers in association with P. falciparum parasitemia in bed net (reduced by 63%) and curtain villages (53%) when compared with controls. Entomologic inoculation rates in both bed net and control villages decreased by more than 50% below control values during both high transmission seasons. The results of this study, together with a 1988 study in the same area during the low transmission season, show that bed nets offer greater year-round of protection against P. falciparum infection than curtains. However, during the high transmission season, this technique reduces the frequency of P. falciparum infection rather than preventing it entirely.

Comparison of two ribosomal DNA-based methods for differentiating members of the Anopheles gambiae complex (Diptera: Culicidae).

Two DNA-based methods, the restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR), were used to identify mosquitoes of the Anopheles gambiae Giles complex collected in Kenya. Field-collected specimens of An. gambiae, An. arabiensis Patton, and An. merus Donity were tested. From a sample of 208 mosquitoes, 181 (87%) were identified by the RFLP method and 205 (99%) were identified by the PCR method. There was complete concordance between the two methods with regard to species identification. PCR assays were simpler, faster, and more reliable than RFLP assays.

Field evaluation of an enzyme-linked immunosorbent assay for estimating the sporozoite rate in Anopheles albimanus.

We have verified for specimens of Anopheles albimanus that an enzyme-linked immunosorbent assay (ELISA) used to assess Plasmodium vivax and P. falciparum sporozoite antigen rates gives results comparable to the salivary gland dissection method for estimating sporozoite rates. For 14,150 adults of An. albimanus, captured at five locations in Guatemala, we report sporozoite antigen rates of 0.03-0.57%, which correlate with the malaria prevalences at the study sites. We also present data that suggest that specimens of An. albimanus for the ELISA can be obtained more efficiently by cattle corral collections than by the human bait capture method.

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.

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.

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.

Microplate assay analysis of the distribution of organophosphate and carbamate resistance in Guatemalan Anopheles albimanus.

Simple microplate assay methods for determining the frequency of insecticide resistance in single mosquitos were used to study the distribution and localization of organophosphate and carbamate resistance in field populations of Anopheles albimanus Weidemann in Guatemala, where such resistance, caused by heavy use of agricultural pesticides, has long been assumed to be widespread. Areas of complete susceptibility to organophosphates and carbamates were observed, as well as areas where the resistant phenotypes represented up to 98% of the population. Overall, the resistance levels were lower and more localized than expected. Two mechanisms of resistance were identified by the microassay methods. These were the elevated esterase (nonspecific esterase) and insensitive acetylcholinesterase mechanisms which were selected independently, the former (documented for the first time in Central American anophelines) being predominant. These methods represent a promising new technology for the detection and assessment of resistance and will facilitate improved control strategy decisions.

Isozyme variation in Simulium (Edwardsellum) damnosum s.l. (Diptera: Simuliidae) from Kenya.

Isozyme variation of the Simulium damnosum sibling species complex was studied by cellulose acetate electrophoresis (CAE) from four Kenyan river systems. Two enzymes, PGM and HK, were diagnostic and differentiated the larvae collected in Western and Nyanza provinces from the larvae collected at Mt. Kenya. Allele frequency differences of the enzyme PGI allowed about 75% separation of the geographically distinct populations.

Characterization of Simulium (Edwardsellum) damnosum s.l. populations from six river systems in Kenya by cellulose acetate electrophoresis.

Isoenzyme characterization of the Simulium damnosum Theobald sibling species complex from two widely separated geographical areas in Kenya is presented based on 10 enzymatic loci. Four river systems in Western and Nyanza Provinces, namely, the Yala, Lusumu, Isiukhu and the Nzoia harbouring S. damnosum s.l. were compared among themselves and with S. damnosum s.l. collected from the Thiba and the Nyamindi river systems in the Mt. Kenya area. The two populations were easily separable using PGM, HK and, more than 73% of the time, with PGI. Using the first two enzymatic loci, PGM and HK, all the western Kenya S. damnosum s.l. belong to the same population while those from Mt. Kenya areas belong to a different population. In both geographical zones there was less than 20% qualitative and quantitative polymorphism within infraspecific forms in any given breeding area of S. damnosum s.l. Three enzymes, ME, XDH, and G-6-PDH had isomorphic mobilities for both the Mt. Kenya and western Kenya populations. Four other enzyme/substrate systems tested had no satisfactory resolution as a diagnostic value.