Effects of expressing a maleness gene in Anopheles gambiae cells using baculovirus as a gene delivery tool

Rising global temperatures are expected to increase the spread of infectious diseases as warming allows disease-carrying mosquitoes such as Anopheles to survive and reproduce in new regions. To interrogate mosquito gene function rapidly and at scale, we here establish baculovirus as a gene delivery tool in Anopheles cells. We then use this system to express and study the dominant male sex-determining factor Yob, whose molecular function is unknown.

We engineered plasmids with mosquito promoters spanning different strengths, driving genes-of-interest and selection markers, and used them to generate baculoviruses. An. gambiae Ag55 cells were infected over a range of virus doses and exposure durations. Infection efficiency, cell viability, and proliferation were characterized by flow cytometry. We profiled host and viral transcriptional responses by RNA-seq; assessed Yob chromatin association and localization by CUT&Tag and microscopy; and mapped protein-level changes and interactors by proteomics and immunoprecipitation.

We found that baculovirus infection blocks cell cycle progression and induces pronounced transcriptome changes, while Yob expression adds an additional male-biased signature. Yob localized to the nucleus but did not directly associate with chromatin by CUT&Tag, suggesting that it does not act as a transcription factor. Yob expression increased the abundance of nuclear RNA-metabolism factors, including DDX5-like helicases and CCR4-NOT-complex subunits, and Yob-HA co-immunoprecipitated with RNA-metabolism proteins.

Together, these results demonstrate that the baculovirus system provides a versatile platform for studying mosquito gene biology in vitro. Our results imply that Yob's mode of action is not classical DNA binding; rather, Yob is a small nuclear factor that modulates RNA-metabolism machinery, inducing a male transcriptional state.