Medical and Veterinary Entomology (2007) 21, 265–269 © 2007 The Authors Journal compilation © 2007 The Royal Entomological Society 265 Introduction Over the course of the past century, the Punjab area of Pakistan has undergone major physical changes through the development of the Indus Basin Irrigation System (IBIS), which began in 1860. This system, currently the world ’s largest contiguous irrigated area, covers an area of approximately 16 million ha, representing 20% of the total surface area of Pakistan (Kuper, 1997). Development of the IBIS has been associated with a rise in the groundwater table and the accumulation of salts in surface soil (Kuper, 1997). Although Salinity Control and Reclamation Projects (SCARP) has attempted to reclaim the land for agricul- tural use, it was estimated in the early 1990s that 22% of the area was still waterlogged and 23% was salt-contaminated, such that plant growth was affected (Ahmad & Kutcher, 1992). Clearly, environmental changes on this scale affect local flora and fauna and such effects have indeed been observed, specifi- cally in terms of a change in the local composition of malaria mosquito species (Klinkenberg et al., 2004). The study by Klinkenberg et al. (2004) reported a major shift in vector species composition over the past 30 years, with an increase in abundance of Anopheles stephensi Liston s.l. relative to Anopheles culicifacies Giles s.l. Although both species are malaria vectors in Pakistan, An. culicifacies is thought to be the primary vector, especially in rural areas, whereas An. stephensi is considered to be of secondary importance in rural areas and only partially responsible for urban malaria transmission (Reisen & Boreham, 1982; Mahmood et al., 1984; Mahmood & Macdonald, 1985). However, An. stephensi is an important vector in other parts of its species range, such as the Persian/ Arabian Gulf (Manouchehri et al., 1976; Rao, 1984), Northwest Frontier Province (NWFP), Pakistan (Hewitt et al., 1996; Rowland et al., 1997; Graham et al., 2002) and eastern Afghanistan (Rowland et al., 2002). Furthermore, a study by Rowland et al. (2000) demonstrated that Plasmodium falciparum malaria cases in the NWFP peaked in October, when An. culicifacies had completely disappeared but An. stephensi was still present. Molecular genetic studies of Anopheles stephensi in Pakistan N. ALI 1,2 , J. C. C. HUME 1 , S. K. DADZIE 1 and M. J. DONNELLY 1 1 Vector Group, Liverpool School of Tropical Medicine, Liverpool, U.K. and 2 Department of Zoology, University of Peshawar, Peshawar, Pakistan Abstract. Anopheles stephensi Liston s.l. (Diptera: Culicidae) is one of the major vec- tors of malaria in Pakistan, India, Iran and Afghanistan. In parts of its range this species has shown increases in both relative and absolute abundance in what is hypothesized to be a response to human-mediated environmental change resulting from extensive irriga- tion. We attempted to detect the molecular genetic signatures of this population instabil- ity based on three samples obtained from two villages (149/6R and 111/6R) within an irrigation zone in Punjab Province and from one village (Azakhel) outside the irrigation scheme in Northwest Frontier Province (NWFP), Pakistan, using seven microsatellite loci and 682 basepairs of the mitochondrial CO1 gene. For microsatellite loci, high levels of genetic diversity were observed within populations (mean alleles per locus 10.71–11.57; mean heterozygosity 0.703–0.733). Deviation from Hardy–Weinberg expectations was observed for only two microsatellite loci in 21 tests. No genetic differ- entiation was observed between populations and average pairwise F ST values did not differ significantly from zero for any population pair or either marker system. Tests of population expansion for both mitochondrial and microsatellite loci were inconclusive. Key words. Anopheles stephensi, malaria vector, microsatellite loci, mtDNA, population structure, Pakistan. Correspondence: Dr Martin James Donnelly, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K. Tel.: + 44 151 705 3296; Fax: + 44 151 705 3369; E-mail: m.j.donnelly@liv.ac.uk