Phylogeographic Patterns and Genetic Diversity of Anopheles stephensi: Implications for Global Malaria Transmission

Background: Anopheles stephensi, a primary malaria vector in South Asia, is expanding its geographic range, raising concerns about increased malaria transmission. However, critical aspects of its genetic diversity, population structure, and evolutionary dynamics remain poorly understood in Khyber Pakhtunkhwa (KP), Pakistan, an endemic malaria region where An. stephensi is adapting to urban settings, posing challenges for the development of targeted vector control strategies. This study addresses this gap by analyzing COI, COII (cytochrome oxidase subunit I and II), and ITS2 (internal transcribed spacer 2) sequences from An. stephensi populations in KP and comparing them with global isolates. Additionally, egg morphology analysis was conducted to identify the biological form. Methods: Mosquitoes were collected from malaria-endemic districts (Nowshera, Charsadda, and Peshawar) using ovitraps. Eggs were characterized morphologically, and DNA was extracted for PCR amplification of COI, COII, and ITS2 markers. Sequences from 17 Pakistani isolates, along with global sequences, were analyzed. Phylogenetic relationships, haplotype networks, genetic diversity, and neutrality tests (Tajima's D and Fu's Fs) were assessed. Results: Egg morphology confirmed the mysorensis form (13-15 ridges per egg) in KP. COI sequences clustered into two subclades (Punjab and KP), with >99% similarity to global isolates. COII and ITS2 sequences showed high similarity (99.46-100%) with populations from China, Iran, India, and Brazil, reflecting strong genetic connectivity rather than distinct regional clustering. Haplotype analysis identified six COI, ten COII, and ten ITS2 haplotypes, with Hap_2 (50.7%) and Hap_1 (43.3%) being the most prevalent in COI, Hap_7 (29.4%) in COII, and Hap_3 (80.8%) in ITS2. Population genetic analysis revealed higher COI diversity in Pakistan and India, with moderate diversity in COII. Neutrality tests suggested balancing selection in COI for both countries, while COII and ITS2 indicated population contraction in Iran. Conclusions: The findings reveal strong genetic connectivity within regions (e.g., Pakistan) and differentiation across global populations of An. stephensi, highlighting its potential for further expansion and adaptation. These insights are critical for informing global malaria control strategies, particularly in regions vulnerable to vector invasion.