The effect of exuviae ingestion on lysosomal calcium accumulation and the presence of exosomes in the hepatopancreas of Porcellio scaber.

The hepatopancreas of isopods has major functions in food digestion and storage of carbohydrates and lipids. Also, it stores essential and accumulates xenobiotic metals in lysosomal granules within the two major cell types, the S- and B-cells of the tissue. A µCT study on moulting Porcellio scaber has shown mineral within the hepatopancreas lumen, when the animal has ingested their shed cuticle after moulting, suggesting recycling of mineral from the exuviae. This study aims to reveal if the lysosomal metal containing granules store calcium originating from the ingested exuviae. Therefore, we investigated the effect of cuticle ingestion on the elemental composition of the hepatopancreas granules of P. scaber, using electron probe X-ray microanalysis. For the preservation of diffusible elements, samples were high pressure frozen and freeze substituted in acetone and we used Propane-1,3-diol as a floatation medium for sections. We analyzed S- and B-cells of animals in the postmoult and intermoult stage that have ingested their exuviae and, as a negative control, cells from postmoult animals that have not ingested their exuviae. STEM and TEM were used for the investigation of the ultrastructure. Unexpectedly, the cryo-fixed samples contain numerous extracellular vesicles (exosomes) and many multivesicular bodies containing pro-exosomes. We show a significant increase of calcium, copper, zinc and sulphur within the metal granules upon exuviae ingestion, and, after 9 days, a reduction of calcium and zinc. The results indicate transitory storage of calcium from the exuviae within the metal granules and its subsequent utilization in cuticle mineralization.