miR-281-x Regulates Self-Grooming Behavior in Honeybees via Octopamine Signaling

Varroa destructor is a major ectoparasitic threat to honeybee (Apis mellifera) colonies, compromising individual and colony health by transmitting viral pathogens and suppressing the host immune system. Grooming behavior is a critical defense mechanism against Varroa destructor infestation in honeybees. However, the molecular regulation underlying this behavior remains poorly understood. This study identifies differentially expressed small RNAs associated with self-grooming intensity in weak and strong groomers. Specifically, we demonstrate that miR-281-x negatively regulates octopamine production by directly targeting the tyrosine decarboxylase 2 (Tdc2) gene, modulating the intensity of self-grooming behavior. Knockdown of miR-281-x in weak groomers (MW) and overexpression in strong groomers (MS) alter grooming intensity. RNA interference of Tdc2 in MS bees reduces octopamine levels, which decreases grooming. Pharmacological modulation of octopamine signaling in MW bees increases the time to first grooming response and grooming frequency. We identify a miR-281-x-Tdc2-octopamine pathway regulating self-grooming in A. mellifera, showing that miRNAs play a key role in octopamine signaling and offering new insights into the molecular mechanisms of grooming against Varroa destructor.