Effects of the dinoflagellate parasite Hematodinium sp. on the immune response of its crustacean hosts

Faculty of Natural and Environmental Sciences School of Ocean and Earth Science Doctor of Philosophy Effects of the dinoflagellate parasite Hematodinium sp. on the immune response of its crustacean hosts by Jodi Leigh Hoppes The aim of the present study was to investigate the immune response of two UK crustaceans, the edible crab Cancer pagurus and the Norway lobster Nephrops norvegicus, to the dinoflagellate parasite Hematodinium sp. Hematodinium sp. is an emerging global pathogen affecting a number of commercially important crustacean species. Parasites have been found to cause metabolic disruption, and mortality has generally been associated with a massive decrease in haemocyte numbers. Investigations to date have indicated the potential for the parasite to avoid or suppress the host immune system, as well as to increase the likelihood of secondary infection. Hematodinium-host interactions were examined using a combination of in vitro experiments, gene expression analysis and immune assays with the express purpose of determining if a) parasites were capable of avoiding the host immune response or b) parasites were suppressing the host immune response. In vitro experiments were designed to study the response of individual haemocyte populations from Cancer pagurus to Hematodinium sp. in the absence of confounding factors such as secondary infection. Cell viability and gross morphology of separate hyalinocyte and granulocyte cultures were examined, though no significant differences were identified between challenged and näıve cultures. A differential display technique, used to identify differentially expressed genes between Hematodinium-exposed and näıve cultures, found two novel genes from näıve granulocyte cultures: a prophenoloxidase activating factor (ppaf ) and an ATP-dependent RNA helicase DEAD-box protein (ddx ). These genes, in addition to the immune-relevant gene for prophenoloxidase (proPO), were analysed for gene expression changes during Hematodinium sp. exposure. No change in gene expression was found for any of the genes, suggesting a lack of parasite recognition or the loss of a crucial humoral recognition component under in vitro conditions. In vivo analysis of immune parameters of N. norvegicus found that, despite dramatically decreased total haemocyte counts, phenoloxidase activity increased in infected individuals. Haemagglutinin titres and intracellular superoxide (SO) anion activity were not significantly different between infected and control animals, however infected animals had much broader variability in SO activity. SO activity was negatively correlated with Hematodinium sp. presence in the haemolymph which suggested a potential mechanism of immune suppression via SO inhibition. In vivo gene expression studies used differential display to identify differentially expressed genes in Hematodinium-infected and uninfected N. norvegicus. Novel genes were identified from N. norvegicus and coded for two crustin isoforms (NnCrust1 and NnCrust2 ), a 40S ribosomal protein S12 (NnrpS12 ) and a chaperonin containing TCP1 protein (NnCct7 ). Gene expression analysis of these three genes and the immune-relevant proPO gene indicated that NnrpS12 was significantly down-regulated in infected N. norvegicus and proPO was significantly up-regulated. Results were discussed regarding the evidence of Hematodinium sp. to avoid recognition by the host immune system as well as the implications of actively suppressing host immune response by inhibiting the production of superoxide anions. Further consideration was given to the possibility of secondary infection during parasite infection.