The Weberian apparatus is a novel hearing adaptation that facilitates increased hearing sensitivity in otophysan fishes. The apparatus is a complex system composed of modifications to anterior vertebral elements, the inner ear, and the swim bladder. A critical piece of the system that often receives minor attention are the various ligaments that bridge these three regions. The most famous of the ligaments is the interossicular ligament, which connects the Weberian ossicle chain (scaphium-intercalarium-tripus). Several other ligaments are present, including the suspensor (tripus to parapophysis 4) and the triple ligament (tripus-os suspensorium-tunica externa). Here, by combining diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRμCT) with classic histological methods, we shine new light on the first intercostal ligament (ICL1) and discuss its potential function in relation to the Weberian apparatus. ICL1 is nearly absent from the cypriniform literature, typically only mentioned in a general discussion together with other intercostal ligaments. This study examines the development and structure of ICL1 comparatively with the other definitive Weberian ligaments in the zebrafish (Danio rerio). We provide a comprehensive view of three-dimensional shape, development, and composition to generate hypotheses regarding potential functions of ICL1 within the greater Weberian apparatus. Given new detail presented herein regarding the structure of ICL1, modifications to rib 5 and parapophysis 4 for ICL1 attachment, and the alignment of ICL1 with the os suspensorium, we propose a supportive (anchoring) role of ICL1 to aid in minimizing non-optimal movement of the structures of the fourth vertebra. This addition would focus vibrations anteriorly through the ossicle chain with minimal signal loss in zebrafish and other species with similar Weberian apparatus morphologies. We conclude that ICL1 should be included in future analyses of Weberian apparatus function where ligaments are addressed.
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