Rhogocytes are morphologically distinct cells distributed throughout connective tissues of crustaceans and molluscs. Using light microscopy, rhogocytes of the vetigastropod Megathura crenulata were identified by their ovoid shape, and their cytoplasm filled with spherical inclusions which contained lysosomal enzymes, based on uptake of neutral red and staining with LysoTracker dye. Rhogocytes were most abundant in the digestive gland (2,824 rhogocytes/mm2), followed by the connective tissue layer surrounding the middle and posterior esophagus and intestine (1,431 rhogocytes/mm2, 872 rhogocytes/mm2, and 1,190 rhogocytes/mm2, respectively), and were lowest in abundance in the foot (154 rhogocytes/mm2). At the transmission electron microscopy level, characteristic features of rhogocytes were inclusions showing a variety of electron densities, abundant vesicles, and rough endoplasmic reticulum in the cytoplasm, and regions of plasma membrane folded to produce slits connected by thin diaphragms. Although several functions have been proposed for gastropod rhogocytes, much attention has been focused on their possible role in the synthesis of the respiratory pigment hemocyanin. In M. crenulata, this molecule exists in several isoforms called keyhole limpet hemocyanin (KLH). One isoform, KLH1, is a large didecamer and has been used extensively in studies on vertebrate immunology and cancer therapy. We present four lines of evidence indicating rhogocytes in M. crenulata synthesize KLH1. First, at the transmission electron microscopy (TEM) level, dilated cisternae of RER containing material similar in size and shape to KLH were observed in rhogocytes examined throughout the year. Second, KLH1 mRNA was identified exclusively in tissue samples that contained rhogocytes; no mRNA for KLH1 was identified in samples containing only hemocytes. Third, immunoperoxidase staining with antibodies specific to KLH was localized only to rhogocytes. Fourth, in situ hybridization with a probe specific for M. crenulataKLH1 demonstrated KLH1-specific mRNA was present only in rhogocytes. Identification of the cells responsible for the synthesis of KLH is important because of the clinical significance of this molecule.
- hemocyanin;respiratory pigment;pore cell;gastropod
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