Distribution and characterization of rhogocyte cell types in the mantle tissue of Haliotis laevigata

Molluscan rhogocytes are known to be the only cells able to synthesize hemocyanin that is one of the largest respiratory proteins in nature. However, investigation of rhogocyte cells in vitro is limited due to difficulty in isolating and establishing marine cell culture. The aim of this study was to investigate the nature and distribution of rhogocyte cells of Haliotis laevigata in the mantle tissue with respect to the expression of the two known isoforms of hemocyanin. Rhogocyte cells were identified using immunofluorescence-fluorescence in situ hybridization (IF-FISH) that involved simultaneous staining of localized hemocyanin by a polyclonal antibody while the mRNA was hybridized with FISH probes. The distribution of rhogocyte cells was demonstrated using flow cytometry, followed by cell sorting with fluorescence-activated cell sorter (FACS) and confocal microscope imaging for further characterization. Our results suggested that the mantle tissue is dominated by two distinct populations of rhogocyte cells that synthesize hemocyanin type 1. Observation with confocal microscopy of both populations revealed hemocyanin localization in the periphery of the cell membrane. Cell population with higher antibody signal had irregular and elongated cell morphology with punctate mRNA probe signals. The second population with lower antibody signal had ovoid morphology and wide distribution of mRNA probe signals. We suggest that these populations represent two distinct phases of hemocyanin biosynthesis of a single isoform, which is closely related to Haliotis tuberculata type 1 hemocyanin (HtH1). The knowledge acquired in this study enhances the understanding of the biology of rhogocyte cells and biosynthesis of hemocyanin.