Recognizing Incipient Epizootic Shell Disease Lesions in the Carapace 2 of the American Lobster, Homarus americanus

Causal factors leading to Epizootic Shell Disease (ESD) lesions in American Lobster, H. 20 americanus, are not well understood. We explore the structural and physiological bases for 21 development of ESD from preclinical stages invisible to unaided eye to early visible stages. We present 22 a lobster shell model which develops structural functional vulnerability and suggests plausible routes to 23 ESD. Medallions of carapace cuticle are obtained from carapace fixed with protocols to minimize 24 movement of mineral and macromolecular components. Rapid processing of medallions is used to 25 encourage large sample sizes compatible with environmental surveys. Oneand two-dimensional 26 analytic maps of polished sections of the cuticle, obtained with an Electron Microprobe, describe the 27 composite mineral and polymeric structures. MicroRaman Spectroscopy was used to identify bond 28 properties of phosphates and carbonates as well as signatures of organic structures. The frequency and 29 properties of structures identified can be monitored through the lobster molting cycle using a high 30 throughput application of micro-Computed Tomography (μCT). We observed density differences in 31 the calcite layer, exocuticle and endocuticle and the frequency and structure of CaCO3 structures in the 32 endocuticle and membranous layer of carapace cuticle during chosen stages of the molting cycle. The 33 correlative microscopy and μCT of shell structures allows an improved understanding of the lobster 34 cuticle structure. Detailed structural differences quantified through development and under different 35 environmental conditions can provide insight into causes and vulnerabilities associated with ESD. 36