Pumping Venom: Valvilli Architecture and Implications for Stinger Functionality

The internal structure of the insect stinger plays a central role in venom delivery, yet critical components in this process remain poorly understood. Of these, the valvilli, a pair of articulated structures within the valve chamber that act as ‘flaps’, have often been overlooked or described only superficially. Here, we use high-resolution micro-computed tomography (micro-CT), histological sectioning, and confocal laser scanning microscopy (CLSM) to characterize the fine morphology and material composition of the cuticle of ant valvilli. We report structural differentiation within the valvilli, including sharply delimited material zones that may correspond to distinct mechanical properties. CLSM imaging highlights variation in sclerotization, resilin distribution, and ultrastructure, indicating that the valvilli are not uniform flap-like elements but rather complex, partially deformable structures. Furthermore, comparative micro-CT scans of stingers fixed in different actuation states indicate the involvement of a non-antiphasic pattern of valvilli movement during stinger deployment, challenging the current understanding of valvilli-assisted pumping based on rhythmic, alternating movements, as inferred from European honeybees. These findings offer new anatomical insight into the architecture of the ant stinger and provide a refined morphological basis for future studies of venom delivery mechanisms in Hymenoptera; to also foster our understanding of the complex functionality of micro-scale injection and pumping systems in insects.