Proteomic and biochemical approaches to investigate the\ninteractions between the Eastern Oyster, Crassostrea virginica and the MSX\nparasite, Haplosporidium nelsoni

The host parasite system of the MSX parasite, Haplosporidium nelsoni, in the eastern\noyster, Crassostrea virginica, poses many challenges for experimental study. The\ninteractions that are involved in the development of the typically high prevalence and\nintensities of disease encountered in certain environments have not been characterized .\nThe parasite is not-culturable and its life cycle outside of the oyster host remains\nunknown, as such the field is the only source of infective tissues available for study.\nThe main goals of this research were to first, explore of potential protein targets involved\nin infection; second, investigate the tissues that provide optimal comparative value and\nthird, test methodologies that allow for clear comparative analysis. In order to investigate\nthis host parasite system it was first necessary to develop an experimental design that\nenabled the consistency of samples and reliable diagnosis of their disease state. Arriving\nat an experimental design involved the implementation of several laboratory protocols\nincluding one and two dimensional protein gel electrophoresis, enzymatic assays, the use\nof two diagnostic methods, field sampling and experimental field infection, in order to\ndetermine the most promising approach to describing the host parasite interaction.\nTwo environmental systems in which MSX infects the eastern oyster were studied, the\nBras d’Or lakes, Cape Breton, Nova Scotia, Canada and the York river system of the\nChesapeake Bay, Virginia, USA. Within the Bras d’Or lakes samples, a population of\noysters was discovered in which parasite pressure was present but the manifestation of\ndisease within the tissues of oysters from this population was not found. The\nidentification of a differential response to parasite pressure led to the comparison of\nproteins involved in disease from the three Bras d’Or lakes populations sampled. The\nnew landscape of the parasite’s presence in the Bras d’Or along with the targeting of\nseveral tissues and the comparison of different individuals with different disease states\nhighlighted the need to control for variability in protein profiles.\nThe concentration of further comparative protein analyses to the Chesapeake Bay system\nallowed for the experimental field infection of naïve oysters and thus the comparison of\nproteins from the same individual oyster’s haemolymph over time. Once collected, these\nsamples were grouped based on final infection intensity and the comparison of protein\nprofiles indicated the presence of a protein after exposure to an MSX impacted area. This\ndifferentially expressed protein was identified as actin and was consistently observed in\nthe cell free haemolymph lysates from all intensity classes studied. The comparison of\nproteolytic activity of haemolymph over time and between final infection intensity\nclasses identified a protease present within the initial haemolymph samples (naive\nindividuals) occurring at a significantly higher frequency within those individuals that\nwent on to develop high intensity infections. This finding suggests a protein\ndifferentiation found within the naïve population that impacts the outcome of infection of\nthese individuals. The potential of this protease as a marker for heightened disease\nsusceptibility may provide insight into the overall disease process of H. nelsoni within\nthis host. Enzymatic activity also differed significantly among infection intensity classes,\nXI\nas alkaline phosphatase increased over time within intensity groups, as well as\ncomparatively between infection intensities. The monitoring of this activity may allow\nfor the disease to be tracked more readily in populations through testing of haemolymph\nover time.\nHaving established some technical protocols that aided in clearly displaying constituent\nproteins from oyster tissue samples, the experimental field trial was established to\ncompare the haemolymph of the same individual over time and exposure to MSX. This\nled to the identification of protein, protease and enzymatic changes associated with\ninfection intensity. The success of this approach can aid in further characterization of\ninfection as well as establish important indicators of the point at which disease may\noccur.

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