Chronologically inappropriate morphogenesis (<i>Chinmo</i>) is required for maintenance of larval stages of fall armyworm

Broad complex (Br-C) and eip93F (E93) transcription factors promote insect metamorphosis from larva to pupa and from pupa to adult, respectively. Recently, chronologically inappropriate morphogenesis (Chinmo) has been proposed as a larval specifier in <i>Drosophila melanogaste</i>r. However, whether Chinmo is required for larval maintenance in lepidopteran insects, the underlying mechanisms involved in maintaining the larval stage, and its interactions with the JH signaling pathway are not well understood. Here, we used a binary transgenic CRISPR/Cas9 system to knockout <i>Chinmo</i> and <i>Kr-h1</i> (primary response gene in the JH signaling pathway) in the fall armyworm (FAW). <i>Kr-h1</i> knockout induced premature metamorphosis only after L5 (penultimate), whereas <i>Chinmo</i> and <i>Kr-h1</i> double knockout induced premature metamorphosis in L3. Sequencing and differential gene expression (DEG) analysis of RNA isolated from mutants and single-cell multiome ATAC analysis of <i>Chinmo</i>, <i>Kr-h1</i>, and <i>Chinmo</i> and <i>Kr-h1</i> double knockout Sf9 cells revealed that Chinmo participates in chromatin modifications that prevent the promoter accessibility and expression of metamorphosis promoting genes. These results suggest that Chinmo is a larval specifier that plays a major role in preventing metamorphosis in early larval stages by controlling chromatin accessibility near the promoters of genes such as <i>Br-C</i> and <i>E93</i> required for pupal and adult development.