Female sandflies of the genus Phlebotomus in the Old World are vectors of Leishmania parasites, the causative agent of different clinical forms of leishmaniasis . The disease is endemic in 88 countries in five continents with a total of 350 million people at risk .
The parasites within the Leishmania donovani complex usually invade the macrophages of the liver, spleen and bone marrow causing the severe symptoms of the fatal Visceral Leishmaniasis (VL) [3, 4]; while the parasites of L. major complex which cause Cutaneous Leishmaniasis (CL) invade the subcutaneous reticulo-endothelial system and cause self-healing lesions that leave life-long scars [5, 6].
Phlebotomus papatasi (Scopoli, 1786) is the principal vector of Leishmania major in the Old World . It is a widely distributed species found in variable habitats and associated with a wide range of vertebrate hosts [7, 8]. Due to their poor dispersal capacity, populations of P. papatasi are expected to show some genetic structuring along their geographical range as a result of adaptation to local habitats and limited gene flow . Such genetic variability could play an important role in the epidemiology and clinical manifestations of leishmaniasis since it affects the vectorial capacity of the vector [10, 11].
Previous studies focusing on potential population sub-structuring in P. papatasi used a number of molecular markers and provided contrasting results. For example, isoenzyme analyses clearly separated Western Mediterranean P. papatasi populations from those from eastern countries but, predictably, failed to reveal differences between urban and rural sandfly populations in Morocco . Mitochondrial DNA sequences coding for cytochrome b (mtDNA cyt b) and sequences from the second internal transcribed spacer of the ribosomal DNA (rDNA ITS2) were both used to study the population structure of P. papatasi populations from North Africa and the Mediterranean sub-region countries [13, 14]. The analyses of mtDNA cyt b sequences suggested some level of genetic differentiation among widely separated populations and revealed a pattern of isolation-by distance between populations from Syria, Egypt, Israel/Palestine and Turkey . The ND4 mtDNA region was also used in combination with the rDNA ITS2 to study the population structure of P. papatasi from 18 countries from North Africa, the Mediterranean sub-region, Saudi Arabia and India, but revealed no clear phylogeographic structure between those populations. However, signs of restricted gene flow were found among populations from Iran, Egypt, Syria, Yemen and Turkey .
Microsatellite markers combined with Bayesian statistic analysis were recently used to study the population structure of Phlebotomus papatasi populations in countries from the North-African and the Mediterranean sub-regions. This study confirmed the occurrence of highly significant genetic differentiation between some populations . However, the geographical scale of the study did not allow for detecting possible genetic differentiation at the local level, which may be the most relevant for explaining the observed patterns of variation in epidemiologically relevant traits observed in some regions.
Thus, so far, genetic differentiation among P. papatasi populations could not be demonstrated at the local geographical level, despite evidence suggesting that it may occur. For example, Schmidt and Schmidt (1963) observed marked morphometric variations within the Egyptian populations of P. papatasi suggestive of sub-structuring . Using isoenzyme analysis, Kassem et al. reported the presence of polymorphisms at many isoenzyme loci among populations of P. papatasi from Egypt but no significant genetic differentiation could be detected [17, 18]. It must be stressed that the possibility of genetic differentiation in P. papatasi populations at the local scale has not yet been explored using microsatellite markers which, with their higher mutation rates, should be comparatively much more informative than isoenzymes and other sequence loci .
In Sudan, CL is caused by L. major and transmitted by P. papatasi[20, 21]. Before the 1970s, the disease was confined to the western parts of the country. Thereafter, major epidemics occurred along the River Nile and the disease became endemic in many regions of the country . The usual clinical forms of the disease usually heal spontaneously without the need for medical treatment, a matter which discourages patients from attending the health centres, and therefore obscures the incidence of the disease . Visceral Leishmaniasis (VL) which is caused by L. donovani and transmitted by P. orientalis is known to be endemic in eastern and southern parts of Sudan [4, 23], with few scattered foci along the White Nile and Darfur . Recently, uncommon clinical manifestations of CL that did not heal spontaneously nor responded to usual drugs have been reported . Moreover, L. donovani parasites have been isolated from CL lesions of some patients who contracted the disease in Khartoum State, Central Sudan, with no history of travelling to VL endemic sites . Recent studies have demonstrated the possibility of genetic exchanges between different strains and species of the Leishmania parasites [27–29] which may further complicate the epidemiology of the disease since hybrid parasites may adapt differently to the vector and reservoir hosts .
Because different clinical manifestations and the parasite behaviour may also be related to genetic differentiation or sub-structuring within sandfly vector populations [11, 31], we conducted a population genetic study of P. papatasi populations in Sudan from broad to local geographical scale. This was done using a set of 5 microsatellite markers, especially developed for P. papatasi, and given the paucity of such markers, by testing and using additional markers developed for P. perniciosus a related phlebotomine species .
P. papatasi populations were collected from different localities characterized by the distribution of the atypical CL cases. The level of genetic diversity and genetic differentiation among natural populations of P. papatasi was determined using F
and Bayesian assignments. Identifying potential factors leading to genetic differentiation and structuring in P. papatasi populations might improve our understanding of the epidemiology of the disease and help develop appropriate control strategies.