Fig. 1.

Correlation between the morphological changes of embryo and JH signaling in T. domestica. A–F Morphological pattern of embryo during embryonic development in T. domestica, as observed through scanning electron microscopy (SEM). Ventral (A) and lateral (A’) views of the newly formed germ band containing only the most anterior cephalic and thoracic segments at day 2 after egg laying (ED2). Ventral (B) and lateral (B’) views of the germ band with completed segmentation observed at ED3. Dorsal closure, progressing from posterior to anterior, initiates at ED5 (C), advances at ED6 (D), and concludes at ED7 (E). By ED7, morphogenesis and segmentation of the legs are completed, followed by differentiation and maturation of tissues and organs, including legs, until hatching. The shedding of the first embryonic (E1) cuticle occurs at ED9 (F). G Transcriptional dynamics of key genes involved in JH biosynthesis and signaling in whole eggs of T. domestica. FPKM (Fragments Per Kilobase of exon model per Million mapped fragments, which indicates gene expression level in RNA-seq) values of JHAMT, CYP15A1, Met, and Kr-h1 were obtained from RNA-seq data generated from wild-type eggs collected at ED1, ED3, ED5, ED7.ED9, and ED11, respectively. H Effect of JH III application and knockout (KO) of JHAMT, CYP15A1, and Met on Kr-h1 expression in whole eggs of T. domestica. Relative expression levels of Kr-h1 were detected using qPCR, with the ribosomal protein S26 (RPS26) as the reference gene. JH III (100 ng/egg) was applied at ED6, and treated eggs were collected 12 h post-treatment for qPCR analysis (the acetone treated eggs as controls). Gene-specific gRNAs (400 ng/μL) and Cas9 (300 ng/μL) were injected into the freshly laid eggs within 3 h, and eggs were collected at ED10 for qPCR analysis (the eggs injected with Cas9 as controls). All RNA-seq and qPCR data are presented as the mean ± s.e.m. (n = 3)