Chitin and the Gel Within the Electrosensory Organs of Cartilaginous Fishes

Abstract

In 1678, the Italian physician, Stefano Lorenzini discovered a mysterious set of tubular organs inside an electric ray. The function of these organs, named Ampullae of Lorenzini (AoL), was unknown for almost three centuries until physiological and behavioral studies revealed that they were sensitive to weak electric fields. AoL have been observed in all cartilaginous fishes and are used to detect electric fields emitted by other animals and possibly for navigation using the earth’s magnetic field. They are comprised of open pores that lead into tubular canals filled with a viscous hydrogel and terminate distally in lobed structures containing specialized electrosensory cells. External field stimuli must pass through the AoL’s space-filling gel to reach the distal electrosensory cells, yet we do not understand what role the material plays in the process. Further, some recent studies have documented some of the gel’s constituents and properties, but its complete molecular makeup and physical structure remain unknown. In this dissertation, I present biological, physicochemical, and structural evidence that chitin is a component of AoL gel in diverse cartilaginous fish species. This is surprising because, until the recent discovery of endogenous chitin within several fish and amphibian species, it was thought that the structural polysaccharide was not synthesized by vertebrates. Due to the novelty of this field of study, I carried out diverse experiments to satisfy the burden of proof that chitin is prevalent within the AoL and corresponds with the expression of genes that code for chitin synthesizing enzymes (chitin synthases). Chitin binding histochemical probes localized specifically to the gel inside the AoL and treatment with chitin digesting enzymes (chitinases) subsequently eliminated probe binding completely. In situ hybridization studies on chitin synthase genes revealed an overt spatial correspondence between chitin synthase expression and chitin binding probes in the AoL. Additionally, analytical techniques confirmed that the gel material exhibited characteristic chitin signatures. Using the little skate (Leucoraja erinacea) as my experimental system, I documented the developmental onset of the AoL themselves and the appearance of chitin within AoL gel. Subsequently, I observed some surprising features of developing AoL and discovered a curious population of chitinous structures widespread across the skin of developing little skate embryos. Finally, ever interested in the function of chitin within AoL gel, I worked collaboratively with researchers in the Physics and Materials Sciences departments at UC Merced as well as electrical engineers at UC Santa Cruz to provide a descriptive report of the molecular structure of gel from spotted ratfish (Hydrolagus colliei) specimens. Imaging with atomic force and scanning electron microscopy revealed that ratfish gel is seemingly colloidal in nature – composed of aggregating spherical particles. X-ray scattering analyses, proton conductivity assays, and further imaging studies were carried out to investigate the differences between native gel and gel that had been digested with proteolytic enzymes. With these experiments, I learned that proteins are critical for the maintenance of both the gel’s stiffness and organized polymeric network and upon their removal, the remaining material readily forms crystalline structures similar in morphology to published images of chitin nanocrystals.