Current Research

Visualization of putative ion-transporting epithelia in;Amphibalanus amphitrite;using correlative microscopy: Potential function in osmoregulation and biomineralization

Gohad NV, Dickinson GH, Orihuela B, Rittschof D and Mount AS.

Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.

Thoracican barnacles are a unique suborder of crustaceans typified by their calcified exterior, which provides protection to the sessile juvenile and adult. Biomineralization is mediated by a mantle epithelium that appears to be involved in calcium uptake and the secretion of calcium laden matrix. Larval and adult intertidal Balanomorph barnacles tolerate a wide range of salinities and it is hypothesized that active ion transport is the primary mechanism for osmoregulation. We observed adult Amphibalanus amphitrite producing an electrolyte-rich secretion emanating from the junction of the basis and parietal plates. Further study of this region using silver staining microscopic techniques, verified by scanning electron microscopyenergy dispersive spectroscopy, revealed a chloride ion rich mantle epithelium. A distinctive pattern of silver chloride stained epithelia was revealed in all A. amphitrite life stages. These epithelia were observed to contain mitochondria rich cells in nauplius and cyprid larvae (as shown by DASPMI staining visualized with confocal laser scanning microscopy) and therefore exhibit potential for active ion transport. Rhod-5 N (a low affinity cellular Ca2+ indicator) labeling was also observed in all barnacle life stages, in tissues shown to be chloride positive. We suspect that the observed chloride ion rich epithelia facilitate ionic regulation via active transport, and biomineralization via cellular Ca2+ uptake, storage and mobilization.


Visualization of shell matrix proteins in hemocytes and tissues of the Eastern oyster, Crassostrea virginica.

Johnstone MB, Ellis S, Mount AS.

Department of Biological Sciences, Clemson University, Clemson, South Carolina, USA.

The tissues of the oyster were examined for the presence of shell matrix proteins (SMPs) using a combination of Western, proteomic, and epi-fluorescent microscopy techniques. SMP, including 48 and 55 kDa phosphoproteins, was detected in the epithelial cells of mantle, gill, heart, and adductor muscle and linings of arteries and veins. The 48 kDa SMP circulates continuously within the hemolymph, and is present in the immune system hemocytes. It appears to be secreted from hemocytes on induction of shell repair. We suggest that the 48 and 55 kDa proteins are multifunctional and bridge the process of soft tissue repair and shell formation by mediating cellular activities during immune response as well as interacting with the mineral phase during deposition. (c) 2007 Wiley-Liss, Inc.


TEM immunocytochemistry of a 48 kDa MW organic matrix phosphoprotein produced in the mantle epithelial cells of the Eastern oyster (Crassostrea virginica).

Myers JM, Johnstone MB, Mount AS, Silverman H, Wheeler AP.

Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.

Immunohistochemical TEM of Eastern oyster (Crassostrea virginica) mantle epithelial cells using a polyclonal antibody to a gel purified 48 kDa MW oyster shell phosphoprotein revealed that it is phosphorylated in the Golgi, packaged into secretory vesicles and subsequently exocytosed across the apical membrane of specialized cells. These phosphoprotein producing cells are concentrated along the mantle side facing the shell, in the region of the outer mantle lobe. A layer of calcium enriched immuno-reactive mucous is associated with the apical microvilli of these cells. The 48 kDa phosphoprotein forms a component of the fibrous organic matrix and appears to be involved in calcium supply thus enabling crystal growth at the mineralization front.


Hemocyte-mediated shell mineralization in the eastern oyster.

Mount AS, Wheeler AP, Paradkar RP, Snider D.

Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.

The growth of molluscan shell crystals is usually thought to be initiated from solution by extracellular organic matrix. We report a class of granulocytic hemocytes that may be directly involved in shell crystal production for oysters. On the basis of scanning electron microscopy (SEM) and x-ray microanalysis, these granulocytes contain calcium carbonate crystals, and they increase in abundance relative to other hemocytes following experimentally induced shell regeneration. Hemocytes are observed at the mineralization front using vital fluorescent staining and SEM. Some cells are observed releasing crystals that are subsequently remodeled, thereby at least augmenting matrix-mediated crystal-forming processes in this system.
















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