Structure and functional analysis of two insulin superfamily members in the Pacific white shrimp Litopenaeus vannamei

The insulin superfamily constitutes a phylogenetically conserved hormonal system that critically regulates growth, metabolism, and reproduction in Metazoa. However, the functional diversification and the architecture of its regulatory networks of this superfamily remains poorly characterized in crustaceans. Here, we identify and characterize two little-studied insulin superfamily genes in the Pacific white shrimp Litopenaeus vannamei, gonadulin (LvGon) and insulin-related peptide (LvIRP). LvGon is predominantly expressed in neural tissues (brain and thoracic ganglia), while LvIRP is specifically enriched in the ovary. Notably, both of them show significantly elevated expression during the D3-D4 pre-molt stages of the molting cycle. Integrated RNA interference (RNAi) and transcriptomic analysis revealed that knockdown of either LvGon or LvIRP induces elevated hemolymph glucose levels and retarded somatic growth. Furthermore, silencing of either gene significantly increased cumulative molting rates, accompanied by activated ecdysone signaling and enhanced glycogenolysis, highlighting the central role of insulin signaling in balancing energy allocation and developmental homeostasis. Transcriptomic analysis revealed that LvGon knockdown remodeled lipid metabolism pathways in both the hepatopancreas and ovary, including PPAR, cAMP, and eicosanoid signaling, causing lipid dysregulation and oocyte developmental suppression. Based on integrated RNAi-transcriptomic data, we propose a hierarchical regulatory model in which LvGon functions as an upstream modulator that positively regulates a downstream functional module comprising LvIRP and LvILP1. This study provides mechanistic insight into crustacean insulin signaling organization, offering valuable perspectives on arthropod endocrine evolution and potential molecular targets for the genetic improvement of shrimp growth and reproduction.