Cryptic Diversity and Demographic Expansion of Plasmodium knowlesi Malaria Vectors in Malaysia.

Although Malaysia is considered free of human malaria, there has been a growing number of Plasmodium knowlesi cases. This alarming trend highlighted the need for our understanding of this parasite and its associated vectors, especially considering the role of genetic diversity in the adaptation and evolution among vectors in endemic areas, which is currently a significant knowledge gap in their fundamental biology. Thus, this study aimed to investigate the genetic diversity of Anopheles balabacensis , Anopheles cracens , Anopheles introlatus , and Anopheles latens -the vectors for P. knowlesi malaria in Malaysia. Based on cytochrome c oxidase 1 ( CO1 ) and internal transcribed spacer 2 ( ITS2 ) markers, the genealogic networks of An. latens showed a separation of the haplotypes between Peninsular Malaysia and Malaysia Borneo, forming two distinct clusters. Additionally, the genetic distances between these clusters were high (2.3-5.2% for CO1 ) and (2.3-4.7% for ITS2 ), indicating the likely presence of two distinct species or cryptic species within An. latens . In contrast, no distinct clusters were observed in An. cracens , An. balabacensis , or An. introlatus , implying a lack of pronounced genetic differentiation among their populations. It is worth noting that there were varying levels of polymorphism observed across the different subpopulations, highlighting some levels of genetic variation within these mosquito species. Nevertheless, further analyses revealed that all four species have undergone demographic expansion, suggesting population growth and potential range expansion for these vectors in this region.