Neuropeptide bursicon homodimer's roles in reproduction and innate immunity of the red flour beetle, Tribolium castaneum
Authors:   , Li Jingjing 
Published: October 2023

[EMBARGOED UNTIL 8/1/2024] Bursicon is a cystine knot family neuropeptide, composed of bursicon (burs) and partner of burs (pburs) subunits, and can form heterodimers and homodimers to execute different biological functions. While heterodimers regulate cuticle tanning and wing maturation, homodimers mediate innate immunity, midgut stem cell proliferation and energy homeostasis in Drosophila melanogaster and Aedes aegypti. As a neuropeptide with multiple functions, the full extent of the roles and signaling pathways of bursicon, particularly pburs homodimers, remain to be fully elucidated. In this research, the roles of pburs in regulating reproductive physiology and innate immunity, as well as the mechanism that mediates the expression of pburs were investigated in the red flour beetle, Tribolium castaneum. A recent study showed that bursicon induces the transcript level of vitellogenin (Vg) in the black tiger shrimp, Penaeus monodon. However, the underlying mechanism behind this effect is still unclear. In the first part of this dissertation research, the role of bursicon homodimers was investigated in the reproductive physiology of T. castaneum. Knockdown of burs or pburs in 2-day pupae significantly down-regulated the expression levels of Vg1, Vg2 and Vg receptor (VgR) in the females 3- and 5-day post-adult emergence, resulting in abnormal oocytes with limited Vg content. Knockdown of burs repressed the number of eggs laid and completely inhibited egg hatch, while knockdown of pburs significantly decreased the number of eggs laid, hatch rate and offspring larval size, with these RNAi effects persisting to the next generation. Additionally, knockdown of burs or pburs downregulated insulin-like receptor (InR), protein kinase B (Akt), target of rapamycin (TOR) and ribosomal protein S6 kinase (S6K) genes involved in the insulin/insulin-like signaling and TOR (IIS/TOR) signaling pathway. Injection of recombinant pburs (r-pburs) protein increased the expression levels of JH synthesis gene JH acid methyltransferase and JH receptor genes methoprene-tolerant and Taiman, along with InR, Akt, TOR, S6K, Vg and VgR. Notably, bursicon receptor gene T. castaneum rickets (Tcrk) RNAi inhibited the impact of r-pburs injection on the transcript levels of Vg genes. These findings suggest that bursicon homodimers regulate reproduction in T. castaneum by influencing the transcript levels of Vg coding genes and Vg uptake via Tcrk to mediate the transcript levels of JH and IIS/TOR pathway genes. Next, the role of pburs homodimer in signaling innate immunity in larvae of T. castaneum was investigated. Through transcriptome analysis, a set of immune-related genes were identified that respond to pburs RNAi. Treating larvae with r-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes operates through NF-KB transcription factor Relish. Most importantly, a novel AMP, Tenecin 3-like peptide (Ten3LP), was identified which was regulated by pburs via NF-KB transcription factor Dorsal-related immunity factor (Dif)/Drosal2, but not Relish. Ten3LP RNAi was performed, followed by the synthesis of r-Ten3LP for use in bacterial inhibition assays. The functional characterization of Ten3LP revealed that it specifically targets fungi and Gram-positive bacteria, and its activation occurs via the Toll pathway stimulated by pburs. These findings identify new targets for development of potential antibiotics and perhaps for RNAi based pest management technology. Finally, the mechanism that regulates pburs action in T. castaneum was examined. Transforming growth factor-[beta] (TGF-[beta]) also belongs to cystine knot protein family and plays an essential role in controlling a variety of physiological processes, including immunity, development and reproduction in insects. TGF-[beta] signals work through heterotetrametric complexes of type I and type II dual-specificity kinase receptors. Knocking down Punt, the type II receptor of TGF-[beta] identified in T. castaneum larvae, increased the transcript levels of pburs and pburs-induced AMP genes, compared with the control. Punt RNAi induced Relish activation and enhanced the larval anti-Escherichia coli ability. Furthermore, Punt RNAi in newly formed female pupae resulted in abnormal development of ovarioles and ovaries, leading to decreased egg production and complete inhibition of egg hatch. It is suggested that in the larval stage, Punt serves as the pburs suppresser and immune inhibitor, while in female adults, Punt might coordinate other factors to impact reproduction. In summary, this study highlights the diverse and essential roles played by bursicon homodimers in various biological processes, such as immunity, development, and reproduction in T. castaneum. These findings contribute to a deeper understanding of the mechanisms underlying bursicon homodimer functions, particularly those of pburs homodimers, in the reproductive physiology and innate immunity of T. castaneum. Furthermore, the discovery of previously unexplored molecular targets and immunerelated genes presents promising opportunities for developing innovative antibiotics, as well as RNAi-based pest management strategies. As Hans Boman's research into synthesizing new antibiotics demonstrates, understanding these molecular processes can lead to groundbreaking advancements in combating bacterial infections. Delving deeper into the interactions between bursicon, TGF-[beta], and other signaling pathways may offer additional insights into the intricate regulation of physiological processes in insects. This knowledge, in turn, could potentially give rise to innovative strategies for pest control.