Bacteriocyte‐specific antimicrobial peptides regulate the Rhynchophorus ferrugineus – Nardonella symbiosis and represent novel targets for symbiosis‐based pest control

BACKGROUND Many beetles require tyrosine supplementation from endosymbiotic bacteria for exoskeleton synthesis. Weevils harbor the ancient endosymbiont Nardonella within specialized bacteriocytes, a mutualistic association maintained for >125 million years in which the bacterium exclusively preserves tyrosine biosynthetic capability. The red palm weevil, one of the world's most destructive invasive pests causing widespread devastation to palm industries across continents, depends on its Nardonella endosymbiont for survival. Disrupting this obligate symbiosis represents a promising pest control strategy, yet the molecular mechanisms maintaining host–symbiont homeostasis remain poorly understood. Although antimicrobial peptides (AMPs) have been implicated in symbiont regulation in some insects, their functions in this ancient weevil– Nardonella association remain unknown. RESULTS We identified two novel bacteriocyte‐specific AMPs, RfAMP1 and RfAMP2. Unlike canonical immune AMPs, RfAMPs exhibited expression patterns that paralleled endosymbiont dynamics and responded specifically to symbiont presence rather than pathogenic infections. Both peptides localized to Nardonella membranes and cytoplasm. RNAi‐mediated knockdown of either RfAMP1 or RfAMP2 resulted in endosymbiont escape into midgut tissues and increased endosymbiont abundance within bacteriocytes. At subinhibitory concentrations, RfAMPs significantly increased bacterial membrane permeability. Loss of RfAMP function through RNAi disrupted symbiotic homeostasis, impaired tyrosine biosynthesis and severely compromised larval survival. CONCLUSIONS RfAMPs regulate endosymbiont homeostasis and spatial confinement, essential for tyrosine provisioning and host fitness. These findings reveal molecular mechanisms underlying the ancient weevil– Nardonella mutualism and highlight potential targets for red palm weevil management through symbiosis disruption.