**Background:** Diaphorina citri (Asian citrus psyllid) is a major pest of citrus globally because it transmits Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing (HLB), a devastating and incurable citrus disease. Female D. citri only lay eggs after feeding on flush shoots (new growth), suggesting that nutritional signals from flush shoots trigger reproduction. Previous work by the authors showed that the target of rapamycin (TOR) signaling pathway and its downstream effects on juvenile hormone (JH) and 20-hydroxyecdysone (20E) are involved in this process, but the upstream nutritional sensing mechanism remained unknown. The insulin/insulin-like growth factor signaling (IIS) pathway is a conserved nutrient-sensing pathway known to regulate reproduction in many insects. This study investigates whether IIS acts upstream of TOR to mediate flush shoot-induced oviposition in D. citri.
**Methods:** Two insulin-like peptide genes (DcILP1 and DcILP2) and one insulin receptor gene (DcIR) were identified from D. citri. Sequence analysis and phylogenetic trees were constructed using MEGAX. Mated female D. citri were placed on either flush shoots (within 1 week of budding) or mature leaves (20 days after budding), and expression of DcILP1, DcILP2, and DcIR was measured by RT-qPCR at 1, 3, 5, and 7 days. RNA interference (RNAi) was performed by topical application of dsRNA (800 ng/μL) targeting each gene, with dsEGFP as control. After RNAi, females were placed on flush shoots, and egg numbers were counted. Ovarian morphology was examined microscopically at day 5. Expression of DcRheb (a key TOR pathway gene) and DcVg (vitellogenin) was measured by RT-qPCR at days 1 and 5 post-RNAi. Statistical analysis used Student's t-test.
**Key Results:** Sequence analysis showed DcILP1 and DcILP2 contain conserved SP, B-chain, C-peptide, and A-chain regions with conserved cysteines and dibasic cleavage sites. DcIR has a 4,023 bp open reading frame encoding 1,340 amino acids, with extracellular domains (L1, L2, CR, FNIII-1/2/3) forming a V-shaped structure similar to Drosophila melanogaster IR, and a highly conserved intracellular tyrosine kinase domain. Phylogenetic analysis placed DcIR in cluster II. Expression of DcILP1, DcILP2, and DcIR was significantly higher in females fed on flush shoots vs. mature leaves at most time points. Specifically, DcILP1 expression was 3.49-, 2.91-, and 6.87-fold higher at days 3, 5, and 7, respectively. DcILP2 was 1.56-, 2.50-, 3.04-, and 1.91-fold higher at days 1, 3, 5, and 7. DcIR was 1.94-, 3.74-, and 2.94-fold higher at days 3, 5, and 7. RNAi effectively silenced each gene. Egg production was drastically reduced: control females laid 83.75 ± 12.09 eggs per female, while dsDcILP1-treated females laid only 4.05 ± 1.11, dsDcILP2-treated laid 19.70 ± 4.20, and dsDcIR-treated laid 8.00 ± 3.50 eggs per female. Ovarian development was impaired in all RNAi groups, with oocytes arrested at previtellogenic stages and no vitellogenic oocytes observed, whereas controls had fully developed ovaries with vitellogenic oocytes. Expression of DcRheb was significantly reduced: to 24.73% and 21.46% of control at days 1 and 5 after dsDcILP1; 31.06% and 55.54% after dsDcILP2; 40.41% and 60.49% after dsDcIR. DcVg expression was also dramatically reduced: to 10.02% and 28.41% after dsDcILP1; 4.95% and 10.05% after dsDcILP2; 0.81% and 46.36% after dsDcIR at days 1 and 5, respectively.
**Clinical Implications:** This study establishes that the insulin signaling pathway acts as an upstream nutritional sensor that activates the TOR pathway to regulate vitellogenin expression and ovarian development in D. citri in response to feeding on flush shoots. The near-complete ablation of egg production following RNAi of DcILP1, DcILP2, or DcIR identifies these genes as promising molecular targets for pest control strategies aimed at reducing D. citri populations and HLB transmission. The findings also provide fundamental insight into the conserved role of IIS in linking nutrition to insect reproduction.