Caroline Singer, Yuriani Palomino, Lisa D Brown
Cat fleas (Ctenocephalides felis) transmit several medically important bacterial pathogens, yet the mechanisms regulating antibacterial defense within the flea digestive tract remain poorly understood. In insects, the immune deficiency (IMD) signaling pathway regulates antimicrobial peptide (AMP) production by activating the IκB kinase (IKK) complex and the downstream NF-κB transcription factor Relish. Although IMD signaling contributes to pathogen control in cat fleas, the functional role of IKK-dependent signaling has not been examined. To investigate this, adult cat fleas were fed blood containing the NF-κB inhibitor IKK16. Transcript levels of the AMP defensin were quantified using real-time quantitative PCR, antibacterial activity was assessed using in vitro bacterial growth assays with flea gut-derived extracts, and bacterial persistence was evaluated in vivo following exposure to Serratia marcescens-infected blood. Under blood-feeding conditions, IKK16 treatment reduced defensin transcription and decreased the antibacterial activity of flea gut-derived extracts against both Gram-negative and Gram-positive bacteria, demonstrating that IKK-dependent signaling contributes to basal AMP regulation and antimicrobial function in the flea digestive tract. However, despite reduced antibacterial activity in vitro, modulation of IKK-dependent signaling did not alter S. marcescens infection intensity in vivo. Consistent with this finding, defensin expression was strongly induced during bacterial challenge and no longer differed among treatments. Collectively, these findings demonstrate that IKK-dependent signaling contributes to basal antimicrobial activity in the flea digestive tract, while bacterial control during infection is maintained despite pathway perturbation, highlighting the complexity of immune regulation within arthropod barrier tissues.