Neuropeptide bursicon influences reproductive physiology via the IIS/TOR signaling in Tribolium castaneum

Bursicon is a cystine knot family neuropeptide, composed of bursicon (burs) and partner of bursicon (pburs) subunits. It can form heterodimers or homodimers to execute different biological functions. The heterodimers regulate cuticle sclerotization and wing maturation while homodimers mediate innate immunity and midgut stem cell proliferation. In the present study, we investigated the role of bursicon in the reproductive physiology of the red flour beetle, Tribolium castaneum. Knock-down of burs, pburs or Tcrk in 2-day pupae significantly down-regulated the expression of Vg1, Vg2 and Vg receptor (VgR) genes in the females 3- and 5-day post adult emergence, leading to abnormal oocytes with limited Vg content. Silencing of burs repressed egg production and completely inhibited egg hatch, while silencing of pburs dramatically decreased egg production, hatch rate and offspring larval size, and these RNAi effects persisted to the next generation. Furthermore, knockdown of burs or pburs down-regulated the expression of genes encoding insulin receptor (InR), protein kinase B (Akt), TOR and ribosomal protein S6 kinase (S6K). Most importantly, injection of recombinant pburs protein was able to up-regulate the expression of these genes in the normal females and rescue the expression of Vg and VgR in the pburs RNAi females but failed to rescue Vg and VgR in the Tcrk knockdown females. We infer that bursicon homodimers influence Vg expression via receptor Tcrk, possibly by mediating the expression of the JH and insulin/insulin-like signaling (IIS)/target of rapamycin (TOR) pathway genes, thereby regulating reproduction in T. castaneum.