Medical and Veterinary Entomology (2010) 24, 352–360 doi: 10.1111/j.1365-2915.2010.00893.x Morphometric and molecular differentiation of Phlebotomus (Phlebotomus) sandflies N. K H A L I D 1,2 , D. E L N A I E M 3 , M. A B O U D 4 , F. AL RABBA 1 and F. T R I P E T 2 1 Department of Zoology, Faculty of Science, University of Khartoum, Khartoum, Sudan, 2 Centre for Applied Entomology and Parasitology, Keele University, Keele, U.K., 3 Department of Natural Sciences, University of Maryland Eastern Shore, MD, U.S.A. and 4 Department of Biological Sciences, Faculty of Science, Al-Neelain University, Khartoum, Sudan Abstract. The closely related sandfly species of the subgenus Phlebotomus namely, Phlebotomus papatasi (Scopoli, 1786), Phlebotomus duboscqi Neveu-Lemair, 1906 and Phlebotomus bergeroti Parrot, 1934 (Diptera: Psychodidae), are major vectors of Leishmania major (Kinetoplastida: Trypanosomatidae), the causative agent of cutaneous leishmaniasis in the Old World. Although allopatric in most of their distribution, the three species exist sympatrically in many places in central and eastern Sudan. Males of the three species can be distinguished using morphological characters; however, females are much harder to identify, thus complicating epidemi- ological studies. We carried out a morphometric and a molecular study to determine reliable morphological features and develop a polymerase chain reaction (PCR) assay for distinguishing females of these species. Males and females from each species were collected from sites in Sudan, East Africa and from one site in Mali, West Africa. Males were analysed morphologically and 20 characters and 10 character ratios were used in a stepwise discriminant analysis. This led to the identification of four characters with high discriminant loading scores sufficient for accurate male species identification. Male DNA was then used for the development of a PCR-based species diagnostic based on the second internal transcribed spacer (ITS2) of the ribosomal DNA. A set of four primers was developed to generate fragment sizes that are specific to each species and can reliably identify females as well as hybrid DNA. Both the morphometric and the molecular findings of this study have important applications for studies of the epidemiology of cutaneous leishmaniasis. Key words. Phlebotomus bergeroti, Phlebotomus duboscqi, Phlebotomus papatasi, differentiation, molecular diagnostic, morphometry, PCR. Introduction Sandflies of the subgenus Phlebotomus are the major vectors of Leishmania major, the causative agent of cutaneous leishmani- asis in North Africa and other parts of the Middle East (Killick- Kendrick, 1990; Gebre-Michael et al., 1993). Although males of this subgenus are readily distinguishable by the structure of their genitalia, the corresponding females are hard to identify. This problem poses a major challenge to studies on the Correspondence: Noteila Khalid, Department of Zoology, Faculty of Science, Khartoum College of Medical Sciences, PO Box 10995, Khartoum, Sudan. Tel.: +2499 1 225 0207; Fax: +249 1 5514 5960; E-mail: Noteila@hotmail.com epidemiology of leishmaniasis, especially in locations where species such as Phlebotomus papatasi, Phlebotomus bergeroti and Phlebotomus duboscqi are present sympatrically. A good example of this is in Kassala region of eastern Sudan, where the three species are often captured in the same light trap or in spray-catch collections (D. Elnaiem & M. Aboud, unpublished data, 1999). In this area and in other parts of Sudan, sandflies are identified using the taxonomic keys constructed by Kirk & Lewis (1951), Quate (1964), Abonnenc & Minter (1965) © 2010 The Authors 352 Medical and Veterinary Entomology © 2010 The Royal Entomological Society