Abstract
BACKGROUND: Anopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage although it is very different at the larval stage. Despite the development of the species-specific multiplex PCR assay for the An. funestus group, the genomic DNA of Anopheles longipalpis type C specimens can be amplified with the Anopheles vaneedeni and Anopheles parensis primers from this assay. The standard, species-specific An. funestus group PCR, results in the amplification of two fragments when An. longipalpis type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between An. vaneedeni and An. parensis. Anopheles longipalpis type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the An. funestus group, such as An. funestus, An. funestus-like and An. parensis, are included. The present study provides a multiplex assay that will identify An. longipalpis along with other common members of the African An. funestus group, including Anopheles leesoni. METHODS: A total of 70 specimens from six species (An. funestus, An. funestus-like, An. parensis, Anopheles rivulorum, An. vaneedeni and An. leesoni) in the An. funestus group and An. longipalpis type C from Malawi, Mozambique, South Africa and Zambia were used for the study. A restriction fragment length polymorphism (RFLP) assay was designed based on the DNA sequence information in the GenBank database. RESULTS: The enzyme, EcoRI digested only An. longipalpis type C and An. funestus-like after the species-specific An. funestus group PCR assay. The An. longipalpis and An. funestus-like digestion profiles were characterized by three fragments, 376 bp, 252 bp and 211 bp for An. longipalpis type C and two fragments, 375 bp and 15 bp for An. funestus-like. CONCLUSIONS: An RFLP method for the group was developed that is more accurate and efficient than those used before. Hence, this assay would be useful for field-collected adult specimens to be identified routinely in malaria vector research and control studies.