Dosage differential effects of permethrin impregnated into bednets on pyrethroid resistant and susceptible genotypes of the mosquito Anopheles stephensi

Abstract Effects of bednets impregnated with permethrin 200 mg and 500mg/m² on pyrethroid resistant and susceptible strains of Anopheles stephensi and their F hybrid progeny were studied, using free-flying female mosquitoes of these three genotypes, in a room with a human subject under a polyester net, having one of his arms in contact with the treated netting. Unexpectedly an apparently higher feeding rate, but lower knockdown and mortality rates, of mosquitoes were obtained for each of the three genotypes with the higher concentration of 500mg/m² compared with the lower dose of 200mg/m². At the lower dose there was 100% mortality 24 h after exposure of all three genotypes, suggesting that there would not be selection for resistance at this dose. However, at the higher dose there was significantly higher mortality of the susceptible strain than of the F hybrids, suggesting incomplete recessiveness of this resistance and that there would therefore be effective selection for resistance by this dose.
When female mosquitoes were confined in bioassay cones on treated netting, the resistant strain of An.stephensi showed significantly less irritability (scored as the time until first flight take-off) in response to each dose, as compared with the susceptible strain and F, hybrids. The higher dose provoked more irritation of each genotype; this could explain the greater knockdown and mortality rates of mosquitoes exposed to the lower dose which was less irritating and hence more effectively insecticidal. Thus a dose of 200mg/m² is preferable to 500mg/m² for malaria vector control.     

Reduced susceptibility to permethrin and its relationship to DDT resistance in larvae of Anopheles stephensi

Permethrin selection of DDT resistant Anopheles stephensi Liston mosquito larvae produced a reduction in susceptibility to knockdown (2 h exposure) of 17-fold, but only 1.6-fold to kill (24 h exposure). Genetic analysis, incorporating visible mutant markers, was interpreted as indicating that, through multigenic inheritance, several interacting genetic factors were collectively responsible for reduced larval susceptibility to knockdown. These were maintained together only under selection pressure, as the effect was lost quickly in the absence of selection or with outcrossing. The 30-40-fold DDT-resistance found in the parental strain was barely altered by permethrin selection, suggesting no relationship with the major source of DDT resistance. This was confirmed in single family studies. Some evidence for an additional tolerance to DDT was found to be associated with reduced larval susceptibility to permethrin.     

Laboratory selection for and characteristics of pyrethroid resistance in the malaria vector Anopheles funestus

A laboratory colony of Anopheles funestus Giles (Diptera: Culicidae) was established in 2000 from material collected from southern Mozambique where pyrethroid resistance had been demonstrated in the wild population. A subsample of the colony was selected for pyrethroid resistance using 0.1% lambda-cyhalothrin. Bioassay susceptibility tests in subsequent generations F(2) to F(4) showed increased resistance with each successive generation. Survival of individual mosquitoes fed only on 10% sugar solution, increased with age up to 4 days, but by day 10 had decreased significantly. However, females that had been mated and given bloodmeals showed no such increase in mortality with age. Biochemical analysis of resistant and susceptible individuals showed increased monooxygenase and glutathione S-transferase activity but no significant correlation with age of the mosquitoes.     

Distribution and spread of pyrethroid and DDT resistance among the Anopheles gambiae complex in Tanzania

The development of insecticide resistance is a threat to the control of malaria in Africa. We report the findings of a national survey carried out in Tanzania in 2011 to monitor the susceptibility of malaria vectors to pyrethroid, organophosphate, carbamate and DDT insecticides, and compare these findings with those identified in 2004 and 2010. Standard World Health Organization (WHO) methods were used to detect knock‐down and mortality rates in wild female Anopheles gambiae s.l. (Diptera: Culicidae) collected from 14 sentinel districts. Diagnostic doses of the pyrethroids deltamethrin, lambdacyhalothrin and permethrin, the carbamate propoxur, the organophosphate fenitrothion and the organochlorine DDT were used. Anopheles gambiae s.l. was resistant to permethrin in Muleba, where a mortality rate of 11% [95% confidence interval (CI) 6–19%] was recorded, Muheza (mortality rate of 75%, 95% CI 66–83%), Moshi and Arumeru (mortality rates of 74% in both). Similarly, resistance was reported to lambdacyhalothrin in Muleba, Muheza, Moshi and Arumeru (mortality rates of 31–82%), and to deltamethrin in Muleba, Moshi and Muheza (mortality rates of 28–75%). Resistance to DDT was reported in Muleba. No resistance to the carbamate propoxur or the organophosphate fenitrothion was observed. Anopheles gambiae s.l. is becoming resistant to pyrethoids and DDT in several parts of Tanzania. This has coincided with the scaling up of vector control measures. Resistance may impair the effectiveness of these interventions and therefore demands close monitoring and the adoption of a resistance management strategy.     

Insecticide resistance in the malarial mosquito Anopheles arabiensis and association with the kdr mutation

A colony of Anopheles arabiensis Patton (Diptera: Culicidae) from the Sennar region of Sudan was selected for resistance to dichlorodiphenyltrichloroethane (DDT). Adults from the F-16 generation of the resistant strain were exposed to all four classes of insecticides approved for use in malaria vector control and showed high levels of resistance to them all (24-h mortalities: malathion, 16.7%; bendiocarb, 33.3%; DDT, 12.1%; dieldrin, 0%; deltamethrin, 24.0%; permethrin, 0%). Comparisons between the unselected base colony and the DDT-resistant strain showed elevated glutathione-S-transferase (P<0.05) in both sexes and elevated esterases (P<0.05) in males only. The Leu-Phe mutation in the sodium channel gene was detected by polymerase chain reaction and sequencing, but showed no correlation with the resistant phenotype. These results do not provide any explanation as to why this colony exhibits such widespread resistance and further studies are needed to determine the precise mechanisms involved. The implications for malaria vector control in central Sudan are serious and resistance management (e.g. through the rotational use of different classes of insecticides) is recommended.     

Mechanisms of resistance to DDT and pyrethroids in Patagonian populations of Simulium blackflies

Mixed populations of the pest blackflies Simulium bonaerense Coscarón & Wygodzinsky, S. wolffhuegeli (Enderlein) and S. nigristrigatum Wygodzinsky & Coscarón (Diptera: Simuliidae) are highly resistant to DDT and pyrethroids in the Neuquén Valley, a fruit-growing area of northern Patagonia, Argentina. As these insecticides have not been used for blackfly control, resistance is attributed to exposure to agricultural insecticides. Pre-treatment with the synergist piperonyl butoxide (PBO) reduced both DDT and fenvalerate resistance, indicating that resistance was partly due to monooxygenase inhibition. Pre-treatment with the synergist tribufos to inhibit esterases slightly increased fenvalerate toxicity in the resistant population. Even so, biochemical studies indicated almost three-fold higher esterase activity in the resistant population, compared to the susceptible. Starch gel electrophoresis confirmed higher frequency and staining intensity of esterase electromorphs in the resistant population. Incomplete synergism against metabolic resistance indicates additional involvement of a non-metabolic resistance mechanism, such as target site insensitivity, assumed to be kdr-like in this case. Glutathione S-transferase activities were low and inconsistent, indicating no role in Simulium resistance. Knowing these spectra of insecticide activity and resistance mechanisms facilitates the choice of more effective products for Simulium control and permits better coordination with agrochemical operations.     

Comparative evaluation of carbosulfan- and permethrin-impregnated curtains for preventing house-entry by the malaria vector Anopheles gambiae in Burkina Faso

Pyrethroid-impregnated bednets and curtains are widely employed to reduce the risk of malaria transmission, but pyrethroid-resistance is becoming more prevalent among malaria vector Anopheles mosquitoes (Diptera: Culicidae). As an alternative treatment for curtains, we assessed carbosulfan (a carbamate insecticide) in comparison with permethrin as the standard pyrethroid, against endophilic female mosquitoes of the Anopheles gambiae Giles complex in a village near Ouagadougou, Burkina Faso. The main criterion evaluated was the impact of curtains (hung inside windows, eaves and doorways) on the number of An. gambiae s.l. females active indoors at night. Light-traps were operated overnight (21.00-06.00 hours beside occupied untreated bednets) to sample mosquitoes in houses fitted with net curtains treated with carbosulfan 0.2 g ai/m2 or permethrin 1 g ai/m2 or untreated, compared with houses without curtains. The treated and untreated curtains significantly reduced the numbers of mosquitoes collected indoors, compared with houses without curtains. Carbosulfan-treated curtains had a highly significantly greater effect than permethrin-treated or untreated curtains, the scale of the difference being estimated as three-fold. However, there was no significant difference between the impact of untreated and permethrin-treated curtains on densities of An. gambiae s.l. trapped indoors. Samples of the An. gambiae complex comprised An. arabiensis Patton and both the S- and M-forms of An. gambiae Giles s.s. Susceptibility tests revealed some resistance to DDT and low frequencies of permethrin-resistance, insufficient to explain the poor performance of permethrin on curtains. Among survivors from the diagnostic dosage of permethrin were some specimens of all three members of the An. gambiae complex, but the kdr resistance mechanism was detected only in the S-form of An. gambiae s.s. Questions arising for further investigation include clarification of resistance mechanisms in, and foraging behaviour of, each member of the An. gambiae complex in this situation and the need to decide whether carbosulfan-treated curtains are acceptably safe for use to reduce risks of malaria transmission.     

Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana

Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south-western Ghana and from urban and rural localities in Ogun State, south-west Nigeria. Adult mosquitoes, reared from field-collected larvae, were exposed to the WHO-recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post-exposure. Susceptibility of An. gambiae s.l. to DDT was 94-100% in Ghana and 72-100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97-100% in Ghana, 95-100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr-type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid-impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S-form, whereas the M-form predominated in urban samples. It is suggested that 'urban island' populations of An. arabiensis and of An. gambiae s.s. M-form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.     

Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control inAfrica. Elucidation of underlying resistance mechanisms is crucial to improve the successof future control programs. A positional cloning approach was used to identify genesconferring resistance in the uncharacterised rp2 quantitative trait locus (QTL)previously detected in this vector using F6 advanced intercross lines (AIL). A113 kb BAC clone spanning rp2 was identified and sequenced revealing acluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary toA. gambiae, AfCYP6M1 is triplicated in A. funestus, whileAgCYP6Z2 orthologue is absent. Five hundred and sixty-five new singlenucleotide polymorphisms (SNPs) were identified for genetic mapping from rp2P450s and other genes revealing high genetic polymorphisms with one SNP every36 bp. A significant genotype/phenotype association was detected forrp2 P450s but not for a cluster of cuticular protein genes previouslyassociated with resistance in A. gambiae. QTL mapping using F6 AIL confirms therp2 QTL with an increase logarithm of odds score of 5. Multiplex geneexpression profiling of 15 P450s and other genes around rp2 followed byindividual validation using qRT–PCR indicated a significant overexpression in theresistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3,AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective foldchange of 11.2, 6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 andAfCYP6Z3 identified amino acid changes potentially associated with resistancefurther indicating that these genes are controlling the pyrethroid resistance explained bythe rp2 QTL. The characterisation of this rp2 QTL significantly improvesour understanding of resistance mechanisms in A. funestus.