MORPHOLOGY,SYSTEMATICS,EVOLUTION Morphological and Molecular Investigations of Population Structure of Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) in Tunisia RAJA BOUDABOUS, 1 KAOUTHER JAOUADI, 1,2 AZZEDINE BOUNAMOUS, 2,3 AND HAMOUDA BABBA 1,4 J. Med. Entomol. 49(4): 787Ð793 (2012); DOI: http://dx.doi.org/10.1603/ME11110 ABSTRACT Morphological and molecular characterization of Phlebotomus (Larroussius) pernicio- sus Newstead and Phlebotomus (L.) longicuspis Nitzulescu in Tunisia is reported. Different localities in central and southern Tunisia were sampled. Sand ßies were collected by sticky-paper traps and Center for Disease Control traps. For morphological study of males, the copulatory valves (aedeagi) were examined and the number of coxite hairs was recorded. For molecular analysis, the mitochondrial cytochrome b gene and the cytochrome c oxidase I gene were sequenced to investigate the population structure of P. perniciosus and P. longicuspis. The majority of P. perniciosus samples from southern and some specimens from central Tunisia showed single-pointed aedeagi curved at their apicies that were indistinguishable from the P. longicuspis aedeagi and appeared similar to the atypical morph of P. perniciosus, previously described in northern Morocco. The current study gives evidence of a wide distribution of atypical morphs of P. perniciosus in southern and central Tunisia. KEY WORDS Phlebotomus perniciosus, Phlebotomus longicuspis, morph type, Tunisia Phlebotomine sand ßies (Diptera, Phlebotominae) are the only known natural vectors of Protozoa of the genus Leishmania (Kinetoplastida: Trypanoso- matidae). In the Mediterranean regions, the proven vectors of human visceral (VL), cutaneous (CL), and canine (CanL) leishmaniasis are Phlebotomus spp. In the western Mediterranean, Phlebotomus (Larroussius) perniciosus Newstead and Phleboto- mus (L.) longicuspis Nitzulescu are the proven and suspected vectors, respectively, of zoonotic leish- maniasis caused by Leishmania infantum Nicolle (1908). In Tunisia, the presence of P. pernicicosus has been noted since 1911 in Djerba (Danseco et al. 1968). However, in the south, it is not a very com- mon species (Croset et al. 1978). It shows a distinct preference for humid to semiarid bioclimates. In pre-Saharan area, P. perniciosus is known to be ab- sent and P. longicuspis is the only Larroussisus spe- cies sufÞciently abundant to be suspected of trans- mitting L. infantum (Dereure et al. 1986). In northern Morocco, Benabdennbi and Pesson (1998) reported that none of the male sand ßies captured exhibited the biÞd copulatory valves characteristic of P. perniciosus. Based on the number of coxite hairs, they found two groups of male morphs: typical P. longicuspis and P. longicuspis-like. The isoenzy- matic analysis classiÞed the P. longicuspis-like morphs as P. perniciosus (Benabdennbi et al. 1999). DNA sequence analysis of mitochondrial cyto- chrome b (mtDNA cyt B) (Pesson et al. 2004), conÞrmed this identiÞcation and indicated the pres- ence of three phylogenetic species in the Moroccan Rif: P. perniciosus including typical (PN) and atyp- ical longicuspis-like (PNA) morphs; P. longicuspis sensu stricto and a sibling species of P. longicuspis (LCx). Recently, PNA was identiÞed morphologi- cally in south-western Morocco (Guernaoui et al. 2005) and in Tunisia (Ghrab et al. 2006, Boudabous et al. 2009). This atypical form has not been reported yet from Algeria (Berchi et al. 2007). We report here the use of morphological characters and polymor- phic sequences of mitochondrial DNA by the se- quencing of two markers, the mtDNA cyt B gene and the cytochrome coxidase I (COI) gene, to in- vestigate the population structure of P. perniciosus and P. longicuspis in central and southern Tunisia. The current study gives evidence of a wide distri- bution of atypical morphs of P. perniciosus (PNA) in southern and central of Tunisia. Materials and Methods Sand Fly Collections. For morphological analysis, sand ßies were collected from 23 localities in central 1 Laboratoire de Parasitologie-Mycologie code 99UR/08-05, Fac- ulte ´ de Pharmacie, De ´ partement de Biologie Clinique B, 1 Rue Avi- cenne, 5000 Monastir Tunisie. 2 USC AFSSA VECPAR, Faculte ´ de Pharmacie, Universite ´ de Reims Champagne-Ardenne, Reims France. 3 Laboratoire de Pharmacologie et Phytochimie, Faculte ´ des Sci- ences de Jijel, Alge ´ rie. 4 Corresponding author, e-mail: hamouda.babba@gnet.tn. 0022-2585/12/0787Ð0793$04.00/0  2012 Entomological Society of America Downloaded from https://academic.oup.com/jme/article/49/4/787/880011 by guest on 31 October 2022