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University of Southampton Research Repository Eprints Soton University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON Effects of the dinoflagellate parasite Hematodinium sp. on the immune response of its crustacean hosts by Jodi Leigh Hoppes A thesis submitted in partial fulfillment for the degree of Doctor of Philosophy in the Faculty of Natural and Environmental Sciences School of Ocean and Earth Science November 2011 \Biology has at least 50 more interesting years." James D. Watson UNIVERSITY OF SOUTHAMPTON Abstract 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 dinoflag- ellate 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) par- asites 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 sec- ondary infection. Cell viability and gross morphology of separate hyalinocyte and granulocyte cultures were examined, though no significant differences were identified between challenged and na¨ıve cultures. A differential display technique, used to identify differentially expressed genes between Hematodinium-exposed and na¨ıve cultures, found two novel genes from na¨ıve granulo- cyte 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 prophenolox- idase (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 de- creased total haemocyte counts, phenoloxidase activity increased in infected individuals. Haemag- glutinin 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. Contents Abstract v List of Figures xiii List of Tables xv Declaration of Authorship xvii Acknowledgements xix Abbreviations xx 1 Introduction1 1.1 General Introduction..............................1 1.2 Disease and crustaceans............................2 1.3 Hematodinium spp................................4 1.3.1 Pathology................................ 10 1.3.2 Hematodinium sp. life cycle and transmission............ 13 1.3.3 Diagnostics............................... 16 1.3.4 Effects of Hematodinium sp. on host physiology and biochemistry 18 1.3.5 Hematodinium sp. effects on immunity................ 19 1.4 Crustacean Immunity............................. 21 1.4.1 Pathogen recognition and pathogen associated molecular patterns (PAMPs)................................ 22 1.4.2 Haemocytes and haemopoiesis.................... 24 1.4.3 Clotting and agglutination...................... 27 1.4.4 Phagocytosis, nodule formation and encapsulation......... 28 1.4.5 Respiratory burst and reactive oxygen species (ROS)........ 29 1.4.6 Antimicrobial peptides......................... 30 1.4.7 Melanisation and the prophenoloxidase system........... 32 1.5 Apoptosis.................................... 34 1.6 Cancer pagurus and Nephrops norvegicus .................. 34 1.7 The current study............................... 35 2 Molecular cloning of crustacean and Hematodinium sp. genes for gene expression studies 37 2.1 Introduction................................... 37 vii Contents viii 2.1.1 Immune-related genes of interest................... 37 2.1.1.1 Prophenoloxidase...................... 37 2.1.1.2 Haemopoiesis......................... 40 2.1.2 Quantifying Hematodinium sp. infection............... 41 2.1.3 Reference Genes............................ 41 2.2 Isolation of species-specific mRNA transcripts................ 42 2.2.1 Cancer pagurus, Nephrops norvegicus and Hematodinium sp. col- lection.................................. 42 2.2.2 Haemolymph collection........................ 43 2.2.3 Haemopoietic tissue .......................... 43 2.2.4 RNA Extraction............................ 44 2.2.5 cDNA Synthesis............................ 44 2.2.6 Degenerate Primer Development................... 46 2.2.7 Polymerase Chain Reaction...................... 48 2.2.8 Degenerate PCR Results........................ 49 2.2.9 Cloning and Sequencing........................ 50 2.2.10 Degenerate PCR sequence results and analysis........... 51 2.2.11 Crustacean actin ............................ 51 2.2.12 Crustacean prophenoloxidase ..................... 52 2.2.13 Hematodinium actin (HemAct).................... 54 2.2.14 Hematodinium 18S........................... 54 2.2.15 Crustacean runt ............................ 57 2.2.16 Rapid amplification of 5' and 3' cDNA ends (RACE)........ 59 2.2.16.1 SMART™ RACE protocol.................. 59 2.2.16.2 RACE Gene-specific primer design............. 60 2.3 Complete proPO sequence results....................... 61 2.4 Conclusions................................... 63 3 Quantitative PCR Methodology 65 3.1 Introduction................................... 65 3.2 Sample handling................................ 67 3.2.1 RNA quality and quantity....................... 68 3.2.1.1 DNase treatment....................... 69 3.2.2 cDNA preparation........................... 69 3.3 qPCR Assay Design and Validation...................... 72 3.3.1 Dye and probe technology....................... 72 3.3.1.1 Specific detection...................... 72 3.3.1.2 Non-specific detection.................... 74 3.3.1.3 qPCR Instrument...................... 74 3.4 Primer and amplicon design.......................... 74 3.4.1 qPCR assay optimisation....................... 76 3.4.1.1 Primer check and melt curve analysis........... 76 3.4.1.2 Primer Titration....................... 78 3.4.1.3 Standard curve for assay efficiency............. 80 3.5 Quantification method............................. 80 3.5.1 Relative quantification......................... 80 3.5.2 Statistical analysis of relative expression qPCR data........ 83 3.5.2.1 Reference gene validation.................. 83 3.5.3 Absolute quantification........................ 84 3.6 Summary.................................... 87 Contents ix 4 Expression of immune-related host genes in Cancer pagurus haemo- cytes during in vitro challenge with Hematodinium sp. 89 4.1 Introduction................................... 89 4.1.1 Host-parasite immune interactions.................. 89 4.1.2 In vitro immune studies........................ 91 4.1.3 Crustacean cell culture......................... 93 4.1.4 Aims of exposing C. pagurus haemocytes
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