High-resolution transcriptional profling of Anopheles gambiae spermatogenesis reveals mechanisms of sex chromosome regulation

Although of high priority for the development of genetic tools to control malaria-transmitting mosquitoes, only a few germline-specifc regulatory regions have been characterised to date and the

presence of global regulatory mechanisms, such as dosage compensation and meiotic sex chromosome inactivation (MSCI), are mostly assumed from transcriptomic analyses of reproductive tissues or whole gonads. In such studies, samples include a signifcant portion of somatic tissues inevitably complicating the reconstruction of a defned transcriptional map of gametogenesis. By exploiting recent advances in transgenic technologies and gene editing tools, combined with fuorescence-activated cell sorting and RNA sequencing, we have separated four distinct cell lineages from the Anopheles gambiae male gonads: premeiotic, meiotic (primary and secondary spermatocytes) and postmeiotic. By comparing

the overall expression levels of X-linked and autosomal genes across the four populations, we revealed

a striking transcriptional repression of the X chromosome coincident with the meiotic phase, classifable as MSCI, and highlighted genes that may evade silencing. In addition, chromosome-wide median

expression ratios of the premeiotic population confrmed the absence of dosage compensation in the male germline. Applying diferential expression analysis, we highlighted genes and transcript

isoforms enriched at specifc timepoints and reconstructed the expression dynamics of the main biological processes regulating the key stages of sperm development and maturation. We generated

the frst transcriptomic atlas of A. gambiae spermatogenesis that will expand the available toolbox for the genetic engineering of vector control technologies. We also describe an innovative and

multidimensional approach to isolate specifc cell lineages that can be used for the targeted analysis of other A. gambiae organs or transferred to other medically relevant species and model organisms.