An investigation of the coelomic fluid and coelomocytes in a New Zealand sea urchin (Kina) Evechinus chloroticus

Abstract Sea urchins fulfil a vital role as a simple scientific model organism and as such have remained a well-used tool in the study of embryonic development and immunology. Sea urchins are known to be immunologically complex, at the level of innate immunity, and have been shown to respond to environmental stresses in a manner similar to the immune response in other animals. Most of the studies have been based on species such as Strongylocentrotus purpuratus or Paracentrotus lividus, while New Zealand’s sea urchin, Evechinus chloroticus (commonly known as kina), has yet to be thoroughly studied. Evechinus chloroticus is one of the largest sea urchin species and is valued culturally and commercially for its edible gonad, or roe. Investigating the stress response of E. chloroticus would provide a greater understanding of their immune system, knowledge that could prove beneficial for potential aquaculture projects involving E. chloroticus. Investigating E. chloroticus would help to elucidate the similarities and differences between E. chloroticus and species already studied such as S. purpuratus and P. lividus. To carry out this investigation into E. chloroticus, samples of sea urchin body fluid, the coelomic fluid, and the coelomocyte cell populations that are found within it, were analysed to elucidate their protein content. To ensure that the immune response was activated, subsets of sea urchins were subjected to environmental stress (prolonged exposure to cold) before samples were taken for analysis. One-dimensional and two-dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) were used to generate protein displays, of sea urchin samples, that allowed a visual comparison of the protein differences between samples. Two-dimensional PAGE (large format) was used for comparative analysis of stressed and unstressed samples of cell-free coelomic fluid and total coelomocytes. Spots were selected, from the protein spot displays, for in-gel enzymatic digestion with trypsin and plated for analysis by mass spectrometry (MALDI –TOF). The peptide sequences generated by MALDI-TOF mass spectrometry were searched against online protein databases, SwissProt and NCBI Echinodermata, for homology with known proteins that could provide putative identities for the unknown proteins. This research identified a number of peptide sequences with homology to known proteins. Proteins were identified as or homologous to proteins related to immune response, cell-to-cell adhesion, iron binding, retinol binding, lipid binding, serine/threonine protein kinases, cell cycle regulation, cellular metabolism, DNA/RNA processing, cytoskeletal interaction, clotting and membrane localisation. The proteins that were identified have provided greater insight into many aspects of the sea urchin, Evechinus chloroticus