Lizette L. Koekemoer

Anopheles funestus resistant to pyrethroid insecticides in South Africa

Northern Kwazulu/Natal (KZN) Province of South Africa borders on southern Mozambique, between Swaziland and the Indian Ocean. To control malaria vectors in KZN, houses were sprayed annually with residual DDT 2 g/m2 until 1996 when the treatment changed to deltamethrin 20–25 mg/m2. At Ndumu (27°02′ S, 32°19′ E) the recorded malaria incidence increased more than six‐fold between 1995 and 1999. Entomological surveys during late 1999 found mosquitoes of the Anopheles funestus group (Diptera: Culicidae) resting in sprayed houses in some sectors of Ndumu area. This very endophilic vector of malaria had been eliminated from South Africa by DDT spraying in the 1950s, leaving the less endophilic An. arabiensis Patton as the only vector of known importance in KZN. Deltamethrin‐sprayed houses at Ndumu were checked for insecticide efficacy by bioassay using susceptible An. arabiensis (laboratory‐reared) that demonstrated 100% mortality. Members of the An. funestus group from Ndumu houses (29 males, 116 females) were identified by the rDNA PCR method and four species were found: 74 An. funestus Giles sensu stricto, 34 An. parensis Gillies, seven An. rivulorum Leeson and one An. leesoni Evans. Among An. funestus s.s. females, 5.4% (4/74) were positive for Plasmodium falciparum by ELISA and PCR tests. To test for pyrethroid resistance, mosquito adults were exposed to permethrin discriminating dosage and mortality scored 24 h post‐exposure: survival rates of wild‐caught healthy males were 5/10 An. funestus, 1/9 An. rivulorum and 0/2 An. parensis; survival rates of laboratory‐reared adult progeny from 19 An. funestus females averaged 14% (after 1 h exposure to 1% permethrin 25 : 75 cis : trans on papers in WHO test kits) and 27% (after 30 min in a bottle with 25 μg permethrin 40 : 60 cis : trans). Anopheles funestus families showing > 20% survival in these two resistance test procedures numbered 5/19 and 12/19, respectively. Progeny from 15 of the families were tested on 4% DDT impregnated papers and gave 100% mortality. Finding these proportions of pyrethroid‐resistant An. funestus, associated with a malaria upsurge at Ndumu, has serious implications for malaria vector control operations in southern Africa.

Bioassay and biochemical analyses of insecticide resistance in southern African Anopheles funestus (Diptera: Culicidae).

Anopheles funestus Giles has been implicated as a major malaria vector in sub-Saharan Africa where pyrethroid insecticides are widely used in agriculture and public health. Samples of this species from northern Kwazulu/Natal in South Africa and the Beluluane region of southern Mozambique showed evidence of resistance to pyrethroid insecticides. Insecticide exposure, synergist and biochemical assays conducted on A. funestus suggested that elevated levels of mixed function oxidases were responsible for the detoxification of pyrethroids in resistant mosquitoes in these areas. The data suggested that this mechanism was also conferring cross-resistance to the carbamate insecticide propoxur.

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

Abstract.  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 F2 to F4 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.

Fungal infection counters insecticide resistance in African malaria mosquitoes

The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies.

Cuticle thickening associated with pyrethroid resistance in the major malaria vector Anopheles funestus

BackgroundMalaria in South Africa is primarily transmitted by Anopheles funestus Giles. Resistance to pyrethroid insecticides in An. funestus in northern Kwazulu/Natal, South Africa, and in neighbouring areas of southern Mozambique enabled populations of this species to increase their ranges into areas where pyrethroids were being exclusively used for malaria control. Pyrethroid resistance in southern African An. funestus is primarily conferred by monooxygenase enzyme metabolism. However, selection for this resistance mechanism is likely to have occurred in conjunction with other factors that improve production of the resistance phenotype. A strong candidate is cuticle thickening. This is because thicker cuticles lead to slower rates of insecticide absorption, which is likely to increase the efficiency of metabolic detoxification.ResultsMeasures of mean cuticle thickness in laboratory samples of female An. funestus were obtained using scanning electron microscopy (SEM). These females were drawn from a laboratory colony carrying the pyrethroid resistance phenotype at a stable rate, but not fixed. Prior to cuticle thickness measurements, these samples were characterised as either more or less tolerant to permethrin exposure in one experiment, and either permethrin resistant or susceptible in another experiment. There was a significant and positive correlation between mean cuticle thickness and time to knock down during exposure to permethrin. Mean cuticle thickness was significantly greater in those samples characterised either as more tolerant or resistant to permethrin exposure compared to those characterised as either less tolerant or permethrin susceptible. Further, insecticide susceptible female An. funestus have thicker cuticles than their male counterparts.ConclusionPyrethroid tolerant or resistant An. funestus females are likely to have thicker cuticles than less tolerant or susceptible females, and females generally have thicker cuticles than males. In pyrethroid resistant An. funestus, this increase in cuticle thickness is likely to have developed as an auxiliary to the primary mode of pyrethroid resistance which is based on enzyme-mediated detoxification.

Evidence of multiple pyrethroid resistance mechanisms in the malaria vector Anopheles gambiae sensu stricto from Nigeria

Pyrethroid insecticide resistance in Anopheles gambiae sensu stricto is a major concern to malaria vector control programmes. Resistance is mainly due to target-site insensitivity arising from a single point mutation, often referred to as knockdown resistance (kdr). Metabolic-based resistance mechanisms have also been implicated in pyrethroid resistance in East Africa and are currently being investigated in West Africa. Here we report the co-occurrence of both resistance mechanisms in a population of An. gambiae s.s. from Nigeria. Bioassay, synergist and biochemical analysis carried out on resistant and susceptible strains of An. gambiae s.s. from the same geographical area revealed >50% of the West African kdr mutation in the resistant mosquitoes but <3% in the susceptible mosquitoes. Resistant mosquitoes synergized using pyperonyl butoxide before permethrin exposure showed a significant increase in mortality compared with the non-synergized. Biochemical assays showed an increased level of monooxygenase but not glutathione-S-transferase or esterase activities in the resistant mosquitoes. Microarray analysis using the An. gambiae detox-chip for expression of detoxifying genes showed five over-expressed genes in the resistant strain when compared with the susceptible one. Two of these, CPLC8 and CPLC#, are cuticular genes not implicated in pyrethroid metabolism in An. gambiae s.s, and could constitute a novel set of candidate genes that warrant further investigation.

Over expression of a Cytochrome P450 (CYP6P9) in a Major African Malaria Vector, Anopheles Funestus, Resistant to Pyrethroids

Anopheles funestus Giles is one of the major African malaria vectors. It has previously been implicated in a major outbreak of malaria in KwaZulu/Natal, South Africa, during the period 1996 to 2000. The re‐emergence of this vector was associated with monooxygenase‐based resistance to pyrethroid insecticides. We have identified a gene from the monooxygenase CYP6 family, CYP6P9, which is over expressed in a pyrethroid resistant strain originating from Mozambique. Quantitative Real‐Time PCR shows that this gene is highly over expressed in the egg and adult stages of the resistant strain relative to the susceptible strain but the larval stages showed almost no difference in expression between strains. This gene is genetically linked to a major locus associated with pyrethroid resistance in this A. funestus population.

Independent mutations in the Rdl locus confer dieldrin resistance to Anopheles gambiae and An. arabiensis

Substitutions of a conserved alanine residue in the Rdl locus coding for a γ‐aminobutyric acid (GABA) receptor subunit with serine or glycine confer resistance to dieldrin in various insect species. Here, we show that alanine to glycine substitution in the Rdl locus of the malaria vector, Anopheles gambiae, is genetically linked to resistance to dieldrin. An alanine to serine substitution developed independently in a dieldrin resistant strain of An. arabiensis. An allele‐specific polymerase chain reaction (PCR) assay was able to differentiate dieldrin resistant and susceptible mosquitoes.

Ribosomal DNA internal transcribed spacer (ITS2) sequences differentiate Anopheles funestus and An. rivulorum, and uncover a cryptic taxon

Differentiation among the closely related Afrotropical species comprising the Funestus Group is difficult by traditional taxonomic measures. Anopheles rivulorum is the second most abundant and widespread species in the Funestus Group, and is occasionally collected indoors along with the dominant member and major malaria vector, An. funestus. The prospect of misidentification of An. rivulorum as An. funestus prompted the development of a rapid, polymerase chain reaction (PCR)‐based method for identifying these two species. The ribosomal internal transcribed spacer 2 (ITS2) was amplified from thirty‐five specimens of An. rivulorum collected from the extremes of its range: Eastern Africa (Kenya), Southern Africa (South Africa) and Western Africa (Burkina Faso). The ITS2 region of An. rivulorum (≈ 380 bp) is sufficiently different in size from the ITS2 of An. funestus (≈ 700 bp) that these species can be distinguished by agarose gel electrophoresis of PCR products without further manipulation. Comparison of the An. rivulorum and An. funestus ITS2 nucleotide sequences revealed such extensive divergence that meaningful alignment was impossible, except for a 25 bp island near the 5′ end. Intraspecific sequence comparisons revealed no variation among An. rivulorum individuals collected from the same country. However, sequence divergence was 2% between specimens from South Africa and Kenya, and nearly tenfold higher (≈ 19%) between specimens from Burkina Faso and either South Africa or Kenya, an unprecedented level of intraspecific ITS2 divergence in Anopheles. Taken together, these data suggest that the Burkina Faso sample is not An. rivulorum, but rather a cryptic taxon within the Funestus Group.

Insecticide resistance in Anopheles arabiensis (Diptera: Culicidae) from villages in central, northern and south west Ethiopia and detection of kdr mutation

BackgroundAnopheles arabiensis is the major vector of malaria in Ethiopia. Malaria vector control in Ethiopia is based on selective indoor residual spraying using DDT, distribution of long lasting insecticide treated nets and environmental management of larval breeding habitats. DDT and pyrethroid insecticides are neurotoxins and have a similar mode of action on the sodium ion channel of insects. It was therefore necessary to verify the insecticide susceptibility status of An. arabiensis, to better understand the status of cross-resistance between DDT and the pyrethroids in this species as well as to detect a resistant gene.MethodsStandard WHO insecticide susceptibility tests were conducted on adults reared from larval and pupal collections from breeding sites at three villages namely: Sodere in the Rift Valley, Gorgora in the north and Ghibe River Valley in the south west of Ethiopia. The occurrence of cross-resistance between pyrethroids and DDT was determined using a DDT selected laboratory colony originally collected from Gorgora. Phenotypically characterized mosquitoes were tested for the presence of knockdown resistance (kdr) alleles using the standard polymerase chain reaction assay.ResultsAll An. gambiae s.l. specimens assayed by PCR were identified as An. arabiensis. The knockdown and mortality results showed An. arabiensis resistance to DDT in all villages, resistance to deltamethrin and permethrin in the Ghibe River Valley and permethrin resistance in Gorgora. Bioassay susceptibility tests also indicated the presence of cross-resistance between DDT and permethrin, but not between DDT and deltamethrin. The knockdown resistance (kdr) mutation of leucine to phenylalanine in the sodium ion channel gene was detected in populations from Gorgora and the Ghibe River Valley.ConclusionSince An. arabiensis shows high levels of resistance to DDT in all villages tested and varying pyrethroid resistance in Gorgora and the Ghibe River valley, precautionary measures should be taken in future vector control operations. Moreover, the status of resistance in other locations in Ethiopia and the spread of resistant gene (s) should be investigated.