Investigation of coelomic fluid from the sea urchin E. chloroticus

The oceans of the world cover approximately 71% of the surface of the earth and are a source of food and recreation for billions of people. Humans have a vested interest in monitoring the health of the marine environment. The modern world’s oceans and coastlines are at risk from repeated exposure to chemical and physical pollution. Sea urchin species are widely distributed around the world. Sea urchin coelomic fluid contains a number of different coelomocytes, proteins and metabolites that are involved in immune defence. This fluid has become the focus of research around the world trying to identify markers of environmental stress. In this research project, a locally available sea urchin species Evechinus chloroticus, was studied. Sea urchins were maintained at a local aquarium facility. Coelomic fluid was harvested from the animals, the coelomocyte cell population was separated, and the cell types sub-fractionated and analysed. Cell-free coelomic fluid was analysed for protein and chromophore composition. Sea urchins were subjected to stress by injection of bacteria into the coelomic cavity and the coelomic fluid was analysed. It was found that the stress treatment induced a 23% increase in the red spherule coelomocyte population. Protein extracts of coelomocytes were prepared for comparison of non-stressed and stressed animals and large format 2D-PAGE followed by in-gel digest MS identified four proteins that were up-regulated in the stressed animals. Ferritin light chain, a subunit of ferritin, involved in iron sequestration, annexin A4, a mediator of phagosome development, peroxiredoxin 6 involved in removal of reactive oxygen species and profilin, which mediates cytoskeletal rearrangement. Analysis of sub-fractionated phagocyte coelomocytes indicated their potential involvement in immune responses by morphology change from petaloid to filopodial forms. Sea urchins subjected to stress showed a significant increase in chromophores, which corresponded to a 50% increase in echinochrome A. Crystallisation of a major chromophore was attempted, as there was the possibility it was bound to a protein. Crystals were formed but it turned out they contained chromophore dimers that formed complexes with transition metals. Ferritin light chain, annexin A4, peroxiredoxin 6, profilin and echinochrome A are all potential markers of stress. The health of the marine environment could one day be assessed by measuring changes in abundance of these compounds in sea urchin species