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Malaria is one of the most devastating diseases in the World. Over 3 billion people live under the threat of malaria in 24 endemic countries (WHO, 2005)Â and it kills over a million people each year in which the children have majority (Korenromp, 2004). There are about 460 recognized anopheles species in the world, out of which, over 100 acts as a vector for human malaria. In relation to 500,000 malaria cases occurring per annum, about 40% of cases subsist due to Plasmodium falciparum, which is significantly more common 64% in the Sindh Province (WHO, 2005). There are 22 anopheles species reported in Pakistan, among which, two anopheles species, Anopheles culicifacies and Anopheles stephensi are the primary malarial vectors in Pakistan. A. culicifacies is considered to be the grave vector in the rural areas (Covell, 1931; Hick and Majid, 1937; Mahmood et al., 1984; Pervez and Shah, 1989), where as A. stephensi holds the same status in the urban areas (Rehman and Mutalib, 1967; Verardi et al., 2002).
Members of the A. culicifacies complex act as the primary vector of both Plasmodium vivax and P. falciparum (Subbaro et al., 1988). A. stephensi is an important vector in Northwest Frontier Province (NWFP), Pakistan (Hewitt et al., 1996; Rowland et al., 1997; Graham et al., 2002) and eastern Afghanistan (Rowland et al., 2002). The A. stephensi showed enormous bionomic variations, which led it to be subdivided into forms. These forms differed in their vectorial capacity and were distinguished by their egg morphology (Sweet and Rao, 1937; Subbarao et al., 1987, 1988). A. stephensi was found to be single, highly variable species, as no hybrid sterility was observed (Rutledge et al., 1970).
Both primary malaria vectors (A. stephensi and A. culicifacies) are endophilic in their diurnal resting habits and are chiefly zoophilic (Reisen, 1978, Reisen & Milby, 1986). Their breeding site is clean water. Breeding sites include temporary pools, agricultural drains, small irrigation channels, pits puddles and paddy fields (Mahmood and McDonald 1985). The peak biting time is after mid night. The seasonal abundance of primary malaria vectors have been described by number of authors including Reisen, 1978; Reisen et al. 1982; Mahmood and McDonald 1985. The seasonal variation in the population of A. culicifacies depends on climate mainly but this specie is found around through out the year. In the cooler areas like NWFP, Balochistan and Azad Jammu & Kashmir (AJK) the densities exhibit unimodal pattern, occurring from May till September. In Punjab and sindh provinces, this species exhibit bi-modal pattern with two peaks one in April and May and other in August and September. While the A. stephensi shows increase in populations by end march, reaching a peak in April and May and thereafter start falling off rapidly until July. It is found in moderate numbers from September to November. In NWFP, Baluchistan and AJK this species is more prominent from July to September.
For establishing the accurate taxonomic status and phylogenetics of species, nuclear and mitochondrial DNA polymorphism in eukaryotic organisms provides practically infinite opportunities. Some of the molecular level genomic studies involving in vitro amplification of DNA by using the practice of polymerase chain reaction (PCR) (Marinucci et al., 1999; Proft et al., 1999; Wilkerson et al., 1995; Chaudhry et al., 2004).
The DNA probe techniques is not reliable because of its sensitivity both to unequal amounts of target DNA loaded on a membrane and to variation in copy number across the different species, and possibly within the same species from different geographical regions (Krzywinski and Besansky , 2003).
Now a days mitochondrial DNA is one of the most widely used region of the genome for DNA diagnostics. There are so many benefits of using mt. DNA including the small size of mitochondrial genome, its single copy number, lack of introns and maternal inheritance, are some of its features for which it is preferred for DNA diagnostics (Hwang and Kim, 1999; Chaudhry et al., 2006; Chaudhry and Kohli, 2007).
In addition to mt. DNA, rDNA gene cluster is also another choice. In between the gene sequences coding for 18S, 5.8S and 28S rRNA are the non-coding internal transcribed spacers 1 (ITS1) and 2 (ITS2) whose sequences are being used for detecting micro and macro geographic genomic variations between species (Morgen and Blair, 1998; Navajas et al., 1998; Marrelli et al., 1999; Hackett and Missiroli, 1935).
Correct identification of malaria vector is essential for targeted malaria control. The discrimination of members of the A. culicifacies species complex is based on cytogenetic methods and has several limitations. In contrast, the rDNA-PCR approach is widely used in the discrimination of cryptic anopheline species (Collins and Paskewitz, 1996).
Mosquito classification in the subfamily Anophelinae is based on the male tarsal claws, male genital organ, morphology of larvae and pupae, etc. (Christophers, 1933). Taxonomically, Anopheles classification based on the above criteria is complex. Modern
advancements in genome organization studies have helped toward understanding the systematics and evolution of mosquitoes in a better perspective. These studies are performed using several molecular features such as DNA content, chromosomal and
mitochondrial DNA organization, DNA sequences of ITS (Internal Transcribed Spacer) and IGS (intergenic sequences) (Besansky and Collins, 1992; Hill and Crampton, 1994). In recent times, an alternative strategy for malaria vector control is possible by exploiting the gross amount of data available on the genomics and proteomics explaining the observed genetic variability in the vector populations. Not all mosquito-malaria 'combinations' are successful; interactions between the two are under strict genetic control (Collins et al., 1986; Vernick et al., 1989). A detailed understanding of the reasons for the failure of malaria transmission by some anophelines may both highlight the normal mechanisms of successful transmission, and also pave the way for the novel vector control strategies. Such studies are limited due to the inadequate knowledge of the genome structure and complexity of the mosquito species (Severson et al., 1994). It is
highly essential to develop molecular markers that would correspond with susceptibility or refractoriness of anophelinesto malaria parasite infection (Hill and Crampton, 1994). It is well established by various studies that the susceptibility status of anophelines for Plasmodium varies even at the species level (cryptic species) (Nanda et al., 1996). This divergence of transmission potential of malaria between very similar types of Anopheles mosquitoes may be due to differential genetic regulations. Therefore, it is highly imperative to study the pattern of vectorial potential in various Anopheles taxa. To do that, a phylogenetic analysis of various Anopheles taxa, which reflects the evolutionary divergence, is required. Moreover, rearrangement of anophelines to evolutionary closer groups, using the scale of genomic divergence, is necessary to evaluate vector divergence.