SHORT COMMUNICATION Species-Specific Primer for Identification of Anopheles quadriannulatus sp. B (Diptera: Culicidae) from Ethiopia Using a Multiplex Polymerase Chain Reaction Assay MESSAY FETTENE 1 AND EMMANUEL A. TEMU Medical Entomology, Department of Clinical Microbiology and Infectious Diseases, School of Pathology of the National Health Laboratory Services and the University of Witwatersrand, P.O. Box 1038, Johannesburg 2000, South Africa J. Med. Entomol. 40(1): 112Ð115 (2003) ABSTRACT Anopheles quadriannulatus Theobald historically has been reported from southern Africa, Zanzibar islands, and Ethiopia. However, based on evidences of genetic incompatibility between crosses of South African and Ethiopian populations, the population from Ethiopia was recently reported as a distinct species designated as An. quadriannulatus sp. B. An. quadriannulatus sp. A, denoted the southern African population. To distinguish the two populations, the IGS (inter- genic spacer) region of rDNA was sequenced to design a primer speciÞc for An. quadriannulatus sp. B. A cocktail polymerase chain reaction (PCR) involving Anopheles gambiae Giles universal (UN) primer, the new primer and other primers speciÞc for members of the An. gambiae complex produced the expected diagnostic products for the respective species. Using extracted DNA and crushed body parts as sources of template DNA, this assay was reliably used to identify samples of An. quadrian- nulatus sp. B. KEY WORDS Anopheles gambiae, Anopheles quadriannulatus sp. B, PCR assay, Ethiopia TWO MEMBERS OF THE Anopheles gambiae Giles com- plexÑAnopheles arabiensis Patton and Anopheles quadriannulatus Theobald have been described from Ethiopia (White 1974). An. quadriannulatus has a markedly zoophilic behavior and is reported to be of little importance in malaria transmission (White 1974). It has been recorded from Ethiopia, Zanzibar islands, and southern Africa (White 1974; Gillies and Coetzee 1987). Populations of An. quadriannulatus from Ethiopia and southern Africa were reported as a single species (White 1974). Hunt et al. (1998), however, reported that populations of An. quadriannulatus from Ethiopia and South Africa are distinct species based on evi- dence of genetic incompatibility between crosses of populations of An. quadriannulatus from those two countries. Thus, the population of An. quadriannulatus from Ethiopia was designated as An. quadriannulatus sp. B, and the southern African population was named “species A.” The most frequently used method to identify spe- cies of the An. gambiae complex is the polymer chain reaction (PCR) assay (Scott et al. 1993) based on sequence differences of the intergenic spacer (IGS) region of the ribosomal DNA (rDNA). This PCR assay distinguishes Þve species of the An. gambiae complex using a universal (UN) primer and four species-spe- ciÞc primers: gambiae (GA), arabiensis (AR), merus/ melas (ME), and quadriannulatus species A (QD). An. quadriannulatus species B, however, cannot be distin- guished using the standard PCR assay (Hunt et al. 1998). Initial investigations into the development of a PCR assay for discriminating An. quadriannulatus sp. A and B had several drawbacks (Fettene et al. 2002). Dis- crimination could be obtained only after the standard identiÞcation based on Scott et al. (1993) was run and specimens were identiÞed as An. quadriannulatus s.l. Thereafter, DNA was extracted from remaining body parts and a further PCR assay was performed to am- plify a large (900 bp) fragment that so far has been observed in only Ethiopian An. quadriannulatus sp. B. This limits the applicability of the technique when individual mosquitoes from large samples of An. gam- biae s.l. mosquitoes must be identiÞed using small body parts. An additional drawback was the added expense of running two PCR assays. In this paper, we provide a species speciÞc primer that ampliÞes a di- agnostic fragment for An. quadriannulatus sp. B when used in combination with the primers devised by Scott et al. (1993). Materials and Methods Mosquito Collection. This work used desiccated mosquitoes collected by indoor resting and pit trap catches from the villages in the Jimma area, Ethiopia. 1 Jimma University, P.O. Box 378, Jimma, Ethiopia (e-mail: messayg@hotmail.com). 0022-2585/03/0112Ð0115$04.00/0  2003 Entomological Society of America Downloaded from https://academic.oup.com/jme/article/40/1/112/904044 by guest on 05 September 2022