Baculovirus-mediated expression of a maleness gene in female Anopheles gambiae cells [RNA-seq]
Background: Rising global temperatures are expected to increase the burden of infectious diseases, including the expansion of mosquito-borne illnesses into new regions. The Anopheles mosquito, the vector of malaria, is a highly sexually dimorphic species; only females require blood meals for egg production and thus transmit disease. Understanding the molecular basis of sex differences in mosquitos may contribute to the design of novel tools for controlling disease transmission. Here, we established baculovirus as a gene delivery tool in female Anopheles cells to investigate the cellular effects after ectopic expression of Yob, the male-determining gene and upstream regulator of sex-specific pathways. Methods: Ag55 cells were infected with different concentrations of baculovirus and incubated for different timepoints and the effects on the number of EGFP+ and viability were assessed by flow cytometry. Comparison of the transcriptome and proteome of Ag55 cells upon ectopic Yob expression was performed. CUT&Tag and microscopy experiments were done to assess Yob chromatin association and location within the cells. Immunoprecipitation of Yob followed by mass spectrometry was used to understand which proteins Yob interacts with. Results were compared using an empty baculovirus. Results: We found that the proportion of detectable EGFP+ cells after infection is promoter-dependent and that the baculovirus blocks cell cycle progression in infected cells. Modest transcriptome changes occurred upon Yob ectopic expression. Yob is a nuclear protein, but did not directly associate with chromatin by CUT&Tag. Proteomics experiments revealed that it upregulates and stabilizes proteins involved in RNA metabolism. In addition, Yob interacts with RNA helicases whose Drosophila orthologues are implicated in dosage compensation. Conclusions: Together, these results demonstrate that the baculovirus system provides a versatile platform for studying mosquito gene biology in vitro. Overall design: Comparative gene expression profiling analysis of RNA-seq data for Ag55 cells.  Each group has four biological replicates. The samples infected with the empty baculovirus are a negative control for Yob-HA expression.