Prevalence and Distribution of Endosymbionts in Bemisia tabaci Populations from Pakistan: Dominance of Arsenophonus in Indigenous Asia II-1 Population

Bemisia tabaci is a globally destructive agricultural pest and an efficient vector of begomoviruses, which cause recurrent epidemics across South Asia, including cotton leaf curl disease in Pakistan. Increasing evidence shows that bacterial endosymbionts play a central role in shaping whitefly biology, population dynamics, and vector competence. However, the distribution of these symbionts remains poorly resolved in Pakistan, a region where begomoviruses are persistent and widespread. This study investigated the cryptic species diversity, secondary endosymbiont composition and their infection frequency in B. tabaci populations collected from major agricultural regions of Pakistan. A total of 274 adult whiteflies belonging to Asia II-1 (n = 199), MEAM-1 (n = 67), Asia I (n = 7), and Asia II-8 (n = 1) were screened using a symbiont-specific PCR assay for six endosymbionts. The primary endosymbiont Candidatus Portiera aleyrodidarum was detected in all individuals, whereas five secondary endosymbionts (Arsenophonus, Cardinium, Hamiltonella, Wolbachia and Rickettsia) were identified with distinct cryptic species- and region-specific patterns. Notably, Arsenophonus was the most prevalent endosymbiont, occurring in 68% of Asia II-1, 100% of Asia I, and 21% of MEAM-1 individuals, with the highest regional prevalence in Punjab (80%) and Khyber Pakhtunkhwa (77%). Logistic regression analyses confirmed significantly higher infection probabilities in indigenous Asia II-1 populations. Network analysis revealed structured co-occurrence patterns, including strong negative associations between Arsenophonus and Hamiltonella. Phylogenetic analyses revealed close relatedness of Pakistani Arsenophonus strains to those reported from neighboring regions, indicating regional community rather than unique local diversification. The dominance of Arsenophonus in Pakistani whitefly populations is of particular significance, given its role in protecting begomoviruses within the insect vector and its implication in facilitating virus persistence and transmission. This study, for the first time in Pakistan, provides a comprehensive assessment of endosymbiont–cryptic species associations in Pakistani B. tabaci populations and highlights the dominant prevalence of Arsenophonus as a potential key player in local virus vector dynamics.