BdNub Is Essential for Maintaining gut Immunity and Microbiome Homeostasis in Bactrocera dorsalis

SIMPLE SUMMARY: The innate immune system of insects can recognize various pathogens that invade insects and make rapid immune responses. However, excessive immune activation is detrimental to the survival of insects. Nub gene of the OCT/POU family plays an important role in regulating the intestinal IMD pathway. In this study, an important horticultural pest, Bactrocera dorsalis, was adopted to study its high adaptability in complex habitats. Through NCBI database analysis, we found that the BdNub gene of B. dorsalis produced two transcription isoforms, BdNubX1 and BdNubX2. After Gram-negative bacterium Escherichia coli with system infection, the immunoeffector genes of Imd signaling pathway, antimicrobial peptides Diptcin (Dpt), Cecropin (Cec), AttcinA (AttA), AttcinB (AttB) and AttcinC (AttC) were significantly up-regulated. The expression levels of antimicrobial peptide genes Dpt, Cec, AttA, AttB, and AttC were significantly up-regulated at 6 h and 9 h after intestinal infection with the Gram-negative bacterium Providencia rettgeri. RNAi showed that the silencing of the BdNubX1 and BdNubX2 genes could make the gut more sensitive to Providencia rettgeri infection, reduce the survival rate significantly, and cause changes in the gut microbiota’s structure. These results suggest that the maintenance of immune balance plays an important role in B. dorsalis high invasiveness. ABSTRACT: Insects face immune challenges posed by invading and indigenous bacteria. They rely on the immune system to clear these microorganisms. However, the immune response can be harmful to the host. Therefore, fine-tuning the immune response to maintain tissue homeostasis is of great importance to the survival of insects. The Nub gene of the OCT/POU family regulates the intestinal IMD pathway. However, the role of the Nub gene in regulating host microbiota remains unstudied. Here, a combination of bioinformatic tools, RNA interference, and qPCR methods were adopted to study BdNub gene function in Bactrocera dorsalis gut immune system. It’s found that BdNubX1, BdNubX2, and antimicrobial peptides (AMPs), including Diptcin (Dpt), Cecropin (Cec), AttcinA (Att A), AttcinB (Att B) and AttcinC (Att C) are significantly up-regulated in Tephritidae fruit fly Bactrocera dorsalis after gut infection. Silencing BdNubX1 leads to down-regulated AMPs expression, while BdNubX2 RNAi leads to increased expression of AMPs. These results indicate that BdNubX1 is a positive regulatory gene of the IMD pathway, while BdNubX2 negatively regulates IMD pathway activity. Further studies also revealed that BdNubX1 and BdNubX2 are associated with gut microbiota composition, possibly through regulation of IMD pathway activity. Our results prove that the Nub gene is evolutionarily conserved and participates in maintaining gut microbiota homeostasis.