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Development of a Challenge Test for the Blue Mussel, Mytilus Edulis

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Development of a Challenge Test for the Blue Mussel, Mytilus Edulis Faculty of Bioscience Engineering Academic Period 2011 – 2012 Development of a Challenge Test for the Blue Mussel, Mytilus edulis Vyshal Delahaut Promotor: Dr. ir. Nancy Nevejan Tutor: Ph.D Mieke Eggermont Thesis submitted in partial fulfillment of the requirements for the academic degree of Master of Science in Aquaculture Copyright The author and promotor give permission to put this thesis to disposal for consultation and to copy parts of it for personal use. Any other use falls under the limitations of copyright, in particular the obligation to explicitly mention the source when citing parts out of this thesis. Date: Nancy Nevejan Mieke Eggermont Vyshal Delahaut Acknowledgements In de eerste plaats wil ik mijn ouders bedanken. Ze hebben mij altijd gesteund tijdens mijn studies en me steeds geholpen als het nodig was. Then, I want to thank my promotor of this thesis, Nancy Nevejan for securing the supply of my test animals, sharing her knowledge about the topic and correcting my written report. I am also grateful to Mieke for guiding me through the experiments, and especially for her encouraging words and jokes when things were more complicated. I also want to thank Tom Defoird for his very good suggestions during the experiments. In addition, his help and advises regarding the PCRs was very instructive, and I did learn many valuable things from him. I am also thankful to Geert Vandewiele for his instructions about the PCR and related procedures, but also for his good company when I was working in the lab. I also want to thank Tom Baelemans for all the work he did for me during my experiments, but also for his jokes and the nice chats I had with him. And last but not least, I am really thankful to Diem. She was, and still is a big support for me. She thought with me about experiments, helped me in the lab as well, or cooked very good meals when I had a lot of practical work. So, cam on nguoi yeu cua toi! Contents PART I. INTRODUCTION ............................................................................................................................. 1 PART II. LITERATURE STUDY ........................................................................................................................ 3 I. Mussel culture in Europe ............................................................................................................... 3 II. The host: Blue mussel (Mytilus edulis) ......................................................................................... 5 III. Pathogens & associated diseases ................................................................................................. 16 IV. Environment ................................................................................................................................ 20 V. Critical discussion of important related research ........................................................................ 20 PART III. MATERIALS AND METHODS ........................................................................................................ 24 I. Standard materials and methods ..................................................................................................... 24 II. Development of a challenge test for Mytilus edulis ........................................................................ 28 III. Development of a molecular toolbox to study the expression of immune genes ............................ 35 IV. Data analysis ................................................................................................................................... 37 PART IV. RESULTS ................................................................................................................................... 38 I. Development of a challenge test ...................................................................................................... 38 II. Development of a molecular toolbox to study the expression of immune genes ............................ 55 PART V. DISCUSSION ............................................................................................................................. 59 I. Development of a challenge test ...................................................................................................... 59 II. Development of a molecular toolbox to study the expression of immune genes ............................ 66 PART VI. CONCLUSION AND RECOMMENDATIONS FOR THE FUTURE .................................................. 69 PART VII. REFERENCES ............................................................................................................................ 71 PART VIII. APPENDIX ................................................................................................................................ 86 Abstract The aim of this study was to develop a protocol for a standardised challenge test for M. edulis larvae. First, a rearing protocol was tested and was positively evaluated for larval experiments in vivo. A next hurdle to take was to find a suitable pathogen. Potential pathogens were identified from literature research and previous lab experiments. Unfortunately, none of the tested bacterial strains caused high mortalities. Consequently, a completely new approach was decided, whereby adult mussels were challenged by an extended range of candidate pathogens through direct injection in the adductor muscle. By changing the environmental conditions during the challenge test, it became clear that the environment plays an extremely important role in the developmental process of diseases. Physical stress and enrichment of the rearing water with bacterial growth media, enhanced mortality caused by the facultative pathogens. Additionally, first steps directing towards DNA-fingerprinting bacterial strains using an ERIC-PCR were made. Finally, three primer pairs were designed for the immune system related genes lysozyme, mytilin B and defensin. Together with one already existing primer pair, they have been tested in standard PCRs. Two primer pairs were positively evaluated, the two other pairs are probably of use as well, but this needs to be confirmed in future testing. The end of the road towards a protocol for a standardised challenge test for M. edulis is not yet in sight, but important new insights were gained during this thesis, including in the field of host-pathogen interaction. PART I. INTRODUCTION A rising trend in aquaculture production of mussels is seen since the last decade. With a yearly world production level of 1,8 million metric ton (FAO, 2010), the mussel industry is facing a very risky situation since it entirely depends on natural spat collection. 1–3 If the capture of mussel larvae will follow the increasing trend in demand, a negative impact on natural populations can be expected. A reduction in population size for example, can create new ecological niches for other (invasive) species.4,5 The strengthening of the environmental legislation in Europe (e.g. Habitats and Birds directives ((92/48/EEC & 79/409/EEC)) correctly aims at preventing such kind of impacts, but as a consequence traditionally accessible seed collection sites have become more protected.6,7 Secondly, the global problem of ocean acidification is expected to negatively affect growth, shell formation and vulnerability of bivalve larvae, and thereby to reduce the natural populations significantly. 8–11 Taken into account these trends, the traditional 100% dependence on natural spat fall can no longer be considered as a sustainable activity, and solutions/alternatives should be investigated. Local shortages of natural mussel seed (e.g.Chili 2012; The Netherlands 2011; Ireland 2003;…)3,12,13 have just recently been reported and are increasing in number. Nowadays, only handful commercial mussel hatcheries worldwide are able to compensate, at least partly for these shortages. 14 In the history of the industrial aquaculture, a multitude of similar cases exist where sudden seed shortages lead to dramatic ecological and economical situations (e.g. tiger shrimp, Penaeus monodon; the bath sponge, Coscinoderma matthewsi;…).15,16This strengthens the need for finding alternative ways to supply seed to commercial mussel farms. Hatchery production could provide the aquaculture industry with a stable amount of seed every year. In this way it would diminish the exposure to natural variations in supply , and open opportunities for genetic selection in function of the market needs .17,18 These were also the main motives to shift to controlled seed production within hatcheries in other aquaculture sectors (e.g. Atlantic salmon, Salmo salar; Nile tilapia, Oreochromis niloticus; White-leg shrimp, Penaeus vannamei; giant tiger prawn, P. monodon; gilt-head sea bream, Sparus aurata; sea bass, Dicentrachus labrax;green shell mussel, Perna viridis…).19–22 INTRODUCTION 1 The currently existing bivalve hatcheries (e.g. scallops and oysters) encounter mainly disease related problems due to the vulnerable larval stages of mollusks.23,24 Therefore, a good knowledge of host-pathogen interaction is essential.25 Figure I-1 illustrates the multifactor approach of disease management in which host, pathogen and environment are the most important factors. In a healthy system an equilibrium exists between these three factors, while interruptions in this balance might lead to a disease outbreak.25 Figure I-1 The different aspects of the pathogen-host-environment continuum. (after Defoird et al, 2007)26 An indispensable tool to study host-pathogen
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