DEVELOPMENT OF A NOVEL FOULING DETERRENCE STRATEGY BY UNDERSTANDING THE EFECT OF NORADRENALINE ON THE CELLS OF EASTERN OYSTER, Crassostrea virginica AND CYPRIS LARVE OF THE STRIPED BARNACLE Balanus amphitrite
Date of Award
Doctor of Philosophy (PhD)
Mount, Andrew S
Hudson , Joan
Boland , Thomas
Metters , Andrew
The focus of this study was to understand and evaluate the effect of noradrenaline (NA) on two prominent fouling marine invertebrates, the Eastern Oyster, Crassostrea virginica and the Striped Barnacle, Balanus amphitrite for the purpose of developing a novel fouling deterrence strategy.
To understand the effect of NA at a cellular level the immune cells (hemocytes) of C. virginica were chosen as representative molluscan cells. Upon stimulation with 10 micro-molar of noradrenaline ~4% hemocytes of C. virginica labeled positive for the beta-adrenergic receptor (beta-AR). Upon NA stimulation the beta-AR positive cells formed cell-cell synapses and apoptosis was detected in ~50% of the hemocyte population.
NA induced apoptosis was followed from the earliest onset to the terminal stages, from the release of cytochrome-c to the DNA degradation. Electric cell substrate impedance measurements suggested that NA stimulation induced cytoskeletal changes in the hemocytes. SDS PAGE and Western analysis corroborated the presence of beta-AR on NA stimulated hemocytes. Mass spectral analysis of the receptor protein revealed that the putative hemocyte beta-AR has a 30% sequence identity to the human beta-AR.
Effect of NA on the settling behavior of B. amphitrite cypris larvae was evaluated by challenging the larvae with micromolar concentrations of NA ranging from 30-100 micro-molar. The noted searching behavior of cypris larvae was lost after NA challenge and the larvae failed to cement to the substratum. ~70% of the NA treated larvae failed to settle and cement. NA treatment caused a considerable delay in cyprid-adult metamorphosis. Metamorphosed juvenile barnacles were observed 96 hours following NA treatment, whereas the control cyprids metamorphosed within 24-48 hours. In the cyprids-adult metamorphosis remnants of the cyprid stage were observed up to 48 hours of development. Metamorphosed juvenile barnacles appeared normal from the presence of growth increments and beating of cirri. NA challenge seemed to promote metamorphosis to juveniles forgoing the settling behavior and cementation to the substratum.
To assess the applicability of NA as a fouling deterrent, NA molecules were covalently conjugated to HEMA (2-hydroxyethyl methacrylate) and MAA (methacrylic acid) polymer surfaces. Ability of covalently conjugated NA molecules to activate their target adrenoreceptors was assessed by understanding the effect of NA-conjugated polymer surfaces on hemocytes of C. virginica.
NA conjugated polymer surfaces induced apoptosis in the hemocytes of C. virginica. Annexin-V assay confirmed the initiation of apoptosis.
Cytoskeletal structure of the hemocytes adhering to NA-conjugated polymer surfaces displayed pronounced degradation. Apoptotic blebbing of plasma membrane was also observed using scanning electron microscopy. Control polymer surfaces failed to exert any deleterious effects on the hemocytes.
Based on the results obtained in this study a novel fouling deterrence strategy is discussed.
Gohad, Neeraj, "DEVELOPMENT OF A NOVEL FOULING DETERRENCE STRATEGY BY UNDERSTANDING THE EFECT OF NORADRENALINE ON THE CELLS OF EASTERN OYSTER, Crassostrea virginica AND CYPRIS LARVE OF THE STRIPED BARNACLE Balanus amphitrite" (2008). All Dissertations. 221.