Synthetic DNA probes to identify members of the Anopheles gambiae complex and to distinguish the two major vectors of malaria within the complex, An. gambiae s.s. and An. arabiensis.

Abstract

Two cloned DNA sequences, lambda C10 and lambda G12, have been isolated from a female Anopheles gambiae sensu stricto genomic DNA library in lambda EMBL4. The lambda C10 clone hybridized with equal intensity to all five of the six species in the An. gambiae Giles complex tested and was therefore suitable for use as a complex-specific clone. The lambda G12 clone was selected for its ability to distinguish the two major vectors of malaria within the complex, An. gambiae s.s. and An. arabiensis. Use of libraries consisting of only female DNA prevented the isolation of male-specific sequences. Southern blot analysis of the cloned DNA permitted the development of smaller Alu I subclones suitable for sequencing that still retained the original specificities and sensitivities of lambda C10 and lambda G12. Each clone was found to possess a series of repeated sequences in direct tandem array of 92-94 and 68 bases, respectively. A comparison of a number of copies of each of the repetitive sequences within the Alu I subclones enabled the definition of consensus sequences for the repetitive elements in lambda C10 and lambda G12. Based on these consensus sequences, two oligonucleotides of 21 and 23 bases designated pAngsl and pAngss were derived from lambda C10 and lambda G12, respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species specificity as the original clones from which they were derived. The nonradioactive, alkaline phosphatase-labeled pAngsl was able to detect as little as 1 ng of target genomic DNA by chemiluminescent detection in a 5-hr autoradiographic exposure. The pAngss probe could detect 5-10 ng of genomic DNA in similar assays. The new probes exhibit great potential for use in An. gambiae complex species identification because they provide both a means of distinguishing the two major vectors of malaria within the complex and of assessing the quality of squashed mosquito samples by providing a means of standardizing hybridization results.