Pumping Venom: Valvilli Architecture in Diacamma (Hymenoptera: Formicidae) and Implications for Stinger Functionality.

The insect stinger is one of the best-known animal venom-delivery structures. Although its internal structure is crucial for venom delivery, key components remain poorly understood. The valvilli are a pair of articulated structures within the valve chamber that act as 'flaps' and have often been overlooked or described only superficially. Here, using Diacamma nr. indicum as a model system, we combine high-resolution micro-computed tomography (micro-CT), histological sectioning, serial block-face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy (CLSM) to characterise the fine-scale morphology and cuticular material composition of ant valvilli. We report structural differentiation within the valvilli, including distinct cuticular zones that likely correspond to different functions. CLSM imaging particularly highlights variation in sclerotisation, resilin distribution and ultrastructure, indicating that the valvilli are not uniformly flexible elements but rather complex, only partially deformable structures. SBF-SEM of the distal lobe margin corroborates this material differentiation at the ultrastructural level, revealing a laterally continuous sublayer of regularly spaced, circular domains with reduced electron density, consistent with a periodically organised transition in cuticle composition or architecture at the procuticle level. 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 in Hymenoptera. By clarifying how such delivery systems have been fine-tuned over the evolution of a hyper-diverse insect group, they may also inform the design of bio-inspired micro-scale injection and pumping systems.