Morphometrics And Molecular Differentiation Of Phlebotomus (Phlebotomus) Sandflies From Sudan

Abstract

The main objectives of this study were to update the geographical distribution of sandfly species in Sudan; differentiate the closely-related females of the subgenus Phlebotomus Phlebotomus, and to investigate the genetic structure of P. papatasi populations, the main vector of cutaneous leishmaniasis (CL) in Sudan. A total of 5023 adult sandflies was collected from different regions of the Sudan during February/2006 to June /2008 using light traps and spraycatches. The collection sites include Khartoum, Kassala, and Gedaref States; Maleik El- Nasir village, northern Sudan and Laya village, Nuba Mountains, southwestern Sudan. Two subgenera and thirteen species were identified using the morphological keys constructed by Abonnenc and Minter (1965), Quate (1964) and Kirk and Lewis (1951). Sergentomyia antennata was a predominant species collected from all the study sites. P. papatasi was the second prevalent species in Khartoum State, while the three Phlebotomus Phlebotomus species viz P. duboscqi, P. bergeroti and P. papatasi were found sympatrically in Wad Shariefai, Kassala State. A morphometric study was carried out to determine reliable morphological characters that can be used to differentiate the three Phlebotomus Phlebotomus species. The head and the last abdominal segments of both the male and female specimens were slide–mounted and measured. Twenty measured characters and their corresponding ratios were subjected to discriminant analysis. Only four characters for the male specimens (Coxite, Style, Genital funnel, and Ascoid4 lengths), and three for the females (Ascoid4 length, Length of antennal segment A4, and Ratio of Asoid4/ A4) were selected by the discriminant analysis as the most discriminating characters among the three species. Depending on these characters, male X specimens were correctly identified while the female ones showed considerable overlap in most of the characters range. The second transcribed spacer of the ribosomal DNA genes (ITS2) has been amplified from male specimens of the three Phlebotomus Phlebotomus using universal primers. Fixed variations within the ITS2 sequence among the three species were utilized to develop species-specific primers for both P. bergeroti and P. duboscqi. Male DNA was then used for the development of a diagnostic PCR assay utilizing a set of four primers (two universal and two species-specific) that generates fragments of different sizes that are specific to each species and can reliably identify females as well as hybrid DNA. Traditional morphometric and multivariate statistical analysis have been used to analyze the geographical variation among P. papatasi populations collected from four allopatric locations in Sudan (Moyleih, Trais, Sirougia, and Wad Shariefai). The discriminant analysis showed insufficient population differentiation although the four populations showed significant differences in most of the measured characters. However, some trends of biogeographical structuring among the four populations can be recognized by grouping of all Khartoum State populations near each other and at a distance from those from Kassala State. These biogeographical structuring may be correlated with environmental conditions persisting in those areas. A set of seven microsatellite loci was utilized to further investigate the population structure of P. papatasi populations in Sudan. Samples of P. papatasi populations were collected from four localities in Khartoum State which represent different ecological biotopes separated by the River Nile and its tributries. Populations from Wad Shariefai, Kassala State, and Egypt were also included in the analysis to investigate any isolation by distance and the possibility of the presence of different lineages of P. papatasi in Sudan. Genetic analysis revealed little population differentaition and XI continuous gene flow among the studied populations however without any signs of isolation. The high migration rate and lack of interpopulation genetic variation among the Sudanese population was attributed to the continuous human and cattle movement among the studied localities that might help in the dispersal of sandflies.