A ribosomal DNAâ€"polymerase chain reaction (PCR) method has been de veloped for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group ofmorphologically indistinguishable sibling mos quito 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 eitherAn. melasinwestern Africa or An. me/as in eastern and southern Africa. The Anopheles gambiae complex is a group of six morphologically indistinguishable yet genet ically and behaviorally distinct mosquito species that vary dramatically in their importance as vectors ofmalaria in Africa.' 2 As many as three and possibly four species may be sympatric in some regions, and at least two occur in most malaria-endemic areas. Current epidemiologic assumptions about their malaria transmission biology are based on an extensive but scientifi cally compromisedliterature in which the species reported, A. gambiae sensu lato, was often a mix ture of at least two members of this complex. Several methods for identifying the species of individual specimens in this complex have been developed, including allozyme analysis, polytene chromosome banding patterns, high-perfor mance liquid chromatography of cuticular hy drocarbons, and hybridization with DNA probes that are either species-specific or reveal species specific restriction enzyme fragments.37 Only the eace&I fnr evntheci7ina the nlienniitlentirle nrinierc ucM in this work, and D. Mills Hamm for constructing the 529
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.
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