Quantifying Calcium in Shrimp Carapace

In order to determine the calcium content in the carapace of the cephalothoraxes of the diseased and healthy shrimps, the carapace of the same location in cephalothoraxes of the diseased and healthy shrimps was fixed in TEM fixative firstly, subsequently subjected to further fixation with 1% osmium tetroxide, then dehydrated, and embedded in Spurr’s resin and prepared ultrathin sections of 50 nm in thickness. The sections were coated with a thin layer of gold-palladium alloy following the previously reported protocols (Letchumanan et al., 2020 (link); Wagner et al., 2020 (link)), and then examined by using X-Max80 EDS (Oxford Instruments, Oxford, Britain) Field Emission Scanning Electron Microscopy (FESEM) at 20 KV. The specimen embedded in Spurr’s resin was also coated with a thin layer of gold-palladium and then inspected by using the FESEM to conduct energy-dispersive X-ray spectroscopy (EDS) to obtain the data of elemental weight percent (%) in dissociation of the main constituent elements in cephalothorax carapace.