<i>Wolbachia</i> and its pWCP plasmid show differential dynamics during the development of <i>Culex</i> mosquitoes

Mosquitoes are major vectors of pathogens such as arboviruses and parasites, causing significant health impacts each year. <i>Wolbachia</i>, an intracellular bacterium widely distributed among arthropods, represents a promising vector control solution. This bacterium can reduce the transmission of dengue, Zika, and chikungunya arboviruses and manipulate the reproduction of its host through its prophage WO. Although research on the <i>Wolbachia</i> mobilome primarily focuses on WO and the phenotypes it induces, the function of <i>Wolbachia</i> plasmid pWCP, recently discovered and reported to be strikingly conserved worldwide, remains unknown. In this study, we analyzed the presence and abundance of pWCP as well as <i>Wolbachia</i> in two different species of <i>Culex</i> mosquitoes, one of the most widespread genera in the world and a vector of numerous diseases. We compared the relative densities of the bacterium and its mobile genetic element in <i>Culex pipiens molestus</i> and <i>Culex quinquefasciatus</i>, a facultatively autogenous and an anautogenous species, respectively, throughout their development from the larval stage L1 to the adult individual specimen using quantitative Polymerase Chain Reaction (PCR). Our results suggest that 2-5 copies of pWCP occur in <i>Wolbachia</i> cells on average, and the plasmid co-replicates with <i>Wolbachia</i> cells. Moreover, <i>Wolbachia</i> and pWCP exhibit differential levels of abundance at specific development stages throughout the mosquito's life cycle in each species. These findings indicate important, and likely beneficial, roles for the plasmid in the bacterium's biology in different mosquito species as well as complex interaction dynamics between <i>Wolbachia</i> and its host during its life cycle.IMPORTANCEMosquitoes of the <i>Culex</i> genus are critical vectors for numerous diseases, causing significant public health concerns. The intracellular bacterium <i>Wolbachia</i> has emerged as a promising vector control solution due to its ability to interfere with pathogen transmission and manipulate mosquito reproduction. However, unlike the extensively studied WO phage, the biological significance and function of <i>Wolbachia's</i> pWCP plasmid, a recently discovered and strikingly conserved mobile genetic element in <i>Culex</i> species, remain unknown. This study investigates the developmental dynamics of pWCP and <i>Wolbachia</i> in two <i>Culex</i> mosquito species, <i>Culex pipiens molestus</i> and <i>Culex quinquefasciatus</i> across their life cycle. In general, the abundance levels of <i>Wolbachia</i> and the plasmid were found to vary across life stages and differ between the two species. However, a relatively small number of pWCP copies were observed per <i>Wolbachia</i> cell, together with a co-replication of the plasmid with the bacterium for most developmental stages. Altogether, these findings suggest a likely beneficial and non-parasitic role for pWCP in <i>Wolbachia</i>'s biology, which may contribute to the intricate interactions between the bacterium and its mosquito hosts.