Population Structure of the Anopheles arabiensis Patton (Diptera: Culicidae) in Sudan (2004-2008)

Abstract

This study was carried out in four Sudanese States, viz., Gadaref, Kassala, Khartoum, and North State during April 2004- April 2008. The study mainly designed to investigate the molecular structure of the Anopheles arabiensis, in Sudan to elucidate the extent of genetic variation between the forms in sympatric and allopatric areas. Two molecular markers (NADH dehydrogenase subunit 5 gene “ND5” of mtDNA and the intergenic spacer region “IGS” of rDNA) were characterized, sequenced and analyzed. Specific primer was specially designed to differentiate between the genetically different forms of An. arabiensis. The entomological survey of this study recognized the presence of the five species of anopheline mosquitoes obtained from the four study areas. These are An. arabiensis (87%), An. rufipes (9%), An. funestus (3%), An. pharoensis (0.8%), and few individuals of An. nili (0.2%). The present work confirmed that, based on the melanin intensity, An. arabiensis populations exhibits two phenotypic forms, known as “normal” and “melanic” form. These two forms were rare in the dry season (November – June). With the start of the rainy season, the density of both forms increased gradually with the normal form reaching its peak during September, and the melanic form reached its peak on October. However, at least during July and August the rate of increase of the normal form was higher than that of the melanic form. A PCR species-specific identification protocol confirmed that An. arabiensis is the only species of An. gambiae complex collected from the studied sites. This technique has also revealed, for the first time in Sudan, the presence of three members of An. funestus group. These were An. funestus s.s. (29%), An. rivurloum (60%) and relatively low density of An. lessoni (11%). It worth mentioning that these three species were collected indoor during December. Clustering of haplotypes of both genetic markers (ND5, 720 bp; n = 174 (720bp) and (IGS, 530bp; n = 66) of An. arabiensis populations revealed fixed five and two nucleotide substitutions, respectively. Based on the ND5 sequence, the level of genetic differentiation among An. arabiensis forms collected from the four Sudanese States were found in the range (FST = 0.58 to 0.60). Moreover, the analysis of genealogy of the ND5 gene provided further evidence for subdivision of An. arabiensis populations in iii Sudan into two molecular forms. Whereas, IGS region of An. arabiensis populations from Kassala and Khartoum provided FST = 0.34 and 0.31 respectively, and the samples from Gadaref and North State showed negative and low FST (-0.012 -0.062) respectively. Using Tsp RI restriction enzyme and the designed specific primers within IGS sequences a distinction between two forms could be differentiated. These two molecular forms were designated by the present author as ARI and ARII. The frequency of the two molecular forms have been studied based on the designed PCR form-specific identification protocol (n= 584) of An. arabiensis populations across the four Sudanese States. In total, 50% (289/584) were found to belong to ARI and 32% (192/584) as heterozygote, whereas 18% (103/584) as ARII. Kassala area showed the highest ratio for ARII (36%; n=50) and similar data was recorded from EL Moeileh area (30%; n= 25) in Khartoum. The population of An. arabiensis obtained from North State were mostly ARI (n=27) and heterozygote (n=5) and there is no ARII. The results of genetic variability of the two molecular markers (ND5 and IGS) indicated that An. arabiensis is undergoing sub-structuring into two molecular forms. The data showed that ARI form was more abundant in areas with permanent breeding sites (as in Gadaref and North State) where ARII more associated with temporary breeding sites mainly those created by human activities – i.e., around water tanks and farms.