The miR ‐176– DcFoxO axis links C Las infection to juvenile hormone signaling and enhanced fecundity in Diaphorina citri

BACKGROUND: The phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), associated with huanglongbing (HLB) disease, is transmitted by the Asian citrus psyllid, Diaphorina citri. CLas-positive females display enhanced fecundity and increased metabolic demands, a phenotype that may promote both vector population growth and bacterial proliferation. Although recent studies have revealed that CLas-associated reproductive enhancement involves neuroendocrine and neurotransmitter pathways, the transcriptional regulators linking persistent bacterial infection, energy metabolism, juvenile hormone (JH) signaling, and ovarian development remain poorly understood. RESULTS: Here, we identify the forkhead transcription factor DcFoxO as a previously unrecognized regulatory node associated with CLas-induced fecundity enhancement in D. citri. DcFoxO expression was significantly elevated in the ovaries of CLas-positive females during reproductive maturation. RNA interference-mediated silencing of DcFoxO reduced triacylglycerol and glycogen reserves, decreased lipid droplet accumulation, impaired ovarian development, reduced fecundity, and lowered CLas titers in the ovaries. Using dual-luciferase reporter assays, RNA immunoprecipitation (RIP), and microRNA (miRNA) gain-of-function experiments, we demonstrated that miR-176 directly targets the 3' untranslated region of DcFoxO and negatively regulates its expression. Overexpression of miR-176 phenocopied DcFoxO knockdown, suppressing energy reserves, ovarian development, fecundity, and CLas proliferation. Mechanistically, DcFoxO silencing or miR-176 overexpression reduced JH titers and down-regulated core JH-responsive reproductive genes, including DcMet, DcKr-h1, DcVg, and DcVgR. CONCLUSION: These findings reveal a miR-176-DcFoxO-JH regulatory axis that links CLas infection to metabolic reprogramming, ovarian maturation, and enhanced vector fecundity. This study adds a transcriptional layer to the emerging model of CLas-mediated manipulation of D. citri reproduction and identifies potential molecular targets for disrupting vector performance and HLB transmission.