Simultaneous identification of the Anopheles funestus group and Anopheles longipalpis type C by PCR-RFLP

Kwang Shik Choi,Maureen Coetzee,Lizette L Koekemoer

doi:10.1186/1475-2875-9-316

Kwang Shik Choi, Maureen Coetzee + Show 1 more

Open Access

https://doi.org/10.1186/1475-2875-9-316

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Abstract

BackgroundAnopheles 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.MethodsA 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.ResultsThe 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.ConclusionsAn 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.

Highlights

Anopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage it is very different at the larval stage
Two fragments (252 bp and 587 bp) of An. longipalpis type C were amplified after the speciesspecific An. funestus group PCR
The restriction site was identical to the DNA sequence of An. parensis, the specific fragment was not involved in the primer design for the method of Koekemoer et al [11]

Summary

Introduction

Anopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage it is very different at the larval stage. The standard, speciesspecific 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 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. Subsequent studies on the systematics of the group have resulted in a reclassification of the group with An. funestus, An. aruni, An. In addition to the 10 species mentioned above, there are closely related species that are not included in the group because of morphological differences in the adult females - Anopheles longipalpis being one of these [1]. It may be a secondary vector of malaria in areas of sufficiently high endemicity and densities of this species [10]

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