Analysis of gene expression inAedes aegyptisuggests changes in early genetic control of mosquito development

ABSTRACT Aedes aegypti, a critical vector for tropical diseases, poses significant challenges for studying its embryogenesis due to difficulties in removing its rigid chorion and achieving effective fixation for in situ hybridization. Here, we present novel methodologies for fixation, dechorionation, DAPI staining, and in situ hybridization, enabling the detailed analysis of gene expression throughout Ae. aegypti embryogenesis. By synchronizing eggs at various developmental stages (0–72 h), we localized the transcripts of the gap gene mille-pattes (mlpt), the dorsoventral gene cactus (cact), and the pioneer transcription factor (pTF) zelda (zld). In situ hybridization and RT-qPCR analyses revealed that mlpt and cact are maternally expresse d, while zld expression begins zygotically during cellularization and later becomes prominent in neuroblasts. Analysis of previously published transcriptomes suggests that three other pTFs, CLAMP, grainyhead and GAF, are also maternally expressed and may function as pioneer transcription factors during Ae. aegypti embryogenesis. These findings suggest that the transcription factors responsible for genome activation in mosquitoes differ from those in fruit flies, highlighting significant divergence in the genetic regulation of early Dipteran embryogenesis.