The tyrosine hydroxylase regulates organs development via affecting hormones level in Gryllus bimaculatus

Tyrosine hydroxylase (TH) is the initial enzyme in the melanin biosynthesis pathway and catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Extensive research has demonstrated that TH exhibits evolutionarily conserved function in insects, regulating multiple physiological pathways including integumentary repair mechanisms, cuticular sclerotization processes, and melanization in response to pathogenic challenges. However, few studies have addressed the role of TH in Gryllus bimaculatus (G. bimaculatus), a hemimetabolous model insect. In this study, we investigated the in vivo role of GbTH gene by using CRISPR/Cas9 gene-editing tool to generate mosaic mutants (GbTH F₀) and homozygous mutants (GbTH^-/-). The GbTH F₀ mutants displayed less pigmented phenotypes, meanwhile the pronotum became thinner, leading to defects in chirping and motor behaviors. The GbTH^-/- mutants exhibited pleiotropic developmental abnormalities, characterized by complete lethality at the nymphal stage. Phenotypic analysis revealed compromised cuticular integrity with impaired chitin matrix deposition in pre-nymphal exoskeletons, accompanied by significantly enhanced desiccation susceptibility and progressive body mass reduction in mutant nymphs. RNA-sequencing transcriptome analysis indicated that GbTH was involved in regulation the synthesis and coordination of juvenile hormone (JH) and ecdysone (20E). Furthermore, GbTH knockout significantly reduced the expression of the lipid transport protein NPC1b. This suppression led to impaired cholesterol uptake, which consequently disrupted steroid hormone biosynthesis and ultimately compromised key developmental transitions, including molting cycles and metamorphic progression. Collectively, these findings establish that GbTH serves as a master regulatory enzyme for the fine-tuning of developmental processes in hemimetabolic insects, extending beyond its canonical function in cuticular pigmentation regulation.