Proteomic and biochemical approaches to investigate the interactions between the Eastern Oyster, Crassostrea virginica and the MSX parasite, Haplosporidium nelsoni

Abstract

The host parasite system of the MSX parasite, Haplosporidium nelsoni, in the eastern oyster, Crassostrea virginica, poses many challenges for experimental study. The interactions that are involved in the development of the typically high prevalence and intensities of disease encountered in certain environments have not been characterized . The parasite is not-culturable and its life cycle outside of the oyster host remains unknown, as such the field is the only source of infective tissues available for study. The main goals of this research were to first, explore of potential protein targets involved in infection; second, investigate the tissues that provide optimal comparative value and third, test methodologies that allow for clear comparative analysis. In order to investigate this host parasite system it was first necessary to develop an experimental design that enabled the consistency of samples and reliable diagnosis of their disease state. Arriving at an experimental design involved the implementation of several laboratory protocols including one and two dimensional protein gel electrophoresis, enzymatic assays, the use of two diagnostic methods, field sampling and experimental field infection, in order to determine the most promising approach to describing the host parasite interaction. Two environmental systems in which MSX infects the eastern oyster were studied, the Bras d’Or lakes, Cape Breton, Nova Scotia, Canada and the York river system of the Chesapeake Bay, Virginia, USA. Within the Bras d’Or lakes samples, a population of oysters was discovered in which parasite pressure was present but the manifestation of disease within the tissues of oysters from this population was not found. The identification of a differential response to parasite pressure led to the comparison of proteins involved in disease from the three Bras d’Or lakes populations sampled. The new landscape of the parasite’s presence in the Bras d’Or along with the targeting of several tissues and the comparison of different individuals with different disease states highlighted the need to control for variability in protein profiles. The concentration of further comparative protein analyses to the Chesapeake Bay system allowed for the experimental field infection of naïve oysters and thus the comparison of proteins from the same individual oyster’s haemolymph over time. Once collected, these samples were grouped based on final infection intensity and the comparison of protein profiles indicated the presence of a protein after exposure to an MSX impacted area. This differentially expressed protein was identified as actin and was consistently observed in the cell free haemolymph lysates from all intensity classes studied. The comparison of proteolytic activity of haemolymph over time and between final infection intensity classes identified a protease present within the initial haemolymph samples (naive individuals) occurring at a significantly higher frequency within those individuals that went on to develop high intensity infections. This finding suggests a protein differentiation found within the naïve population that impacts the outcome of infection of these individuals. The potential of this protease as a marker for heightened disease susceptibility may provide insight into the overall disease process of H. nelsoni within this host. Enzymatic activity also differed significantly among infection intensity classes, XI as alkaline phosphatase increased over time within intensity groups, as well as comparatively between infection intensities. The monitoring of this activity may allow for the disease to be tracked more readily in populations through testing of haemolymph over time. Having established some technical protocols that aided in clearly displaying constituent proteins from oyster tissue samples, the experimental field trial was established to compare the haemolymph of the same individual over time and exposure to MSX. This led to the identification of protein, protease and enzymatic changes associated with infection intensity. The success of this approach can aid in further characterization of infection as well as establish important indicators of the point at which disease may occur