Faculty of Bioscience Engineering

Academic Period 2011 – 2012

Development of a Challenge Test for the Blue , 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 , 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: (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 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 interactions is a challenge test. This is a controlled experiment where a pathogen, known to cause a significant degree of mortality under standard conditions, is administered to the host. Such a test is not developed for M. edulis yet. The aim of this thesis is to develop a challenge test for the blue mussel. This included also the selection of relevant immune related genes and the design and testing of primers for immune gene expression analysis. The development of a challenge test for M. edulis could be regarded as pioneer-work in which the outcome of each experiment led to the design of the next one. While writing this thesis, an attempt is made to describe these steps so the reader could understand the difficulties encountered, and why certain decisions were taken concerning subsequent experiments.

INTRODUCTION 2

PART II. LITERATURE STUDY

First of all a literature review was performed to acquire an overview of the current knowledge of the biology, anatomy and physiology of the host (M. edulis), of possible pathogens, and finally of some environmental aspects (Figure I-1). Additionally, a critical discussion of important related research was performed.

I. Mussel culture in Europe

There are three Mytilus species which naturally thrive in the European coastal waters; Mytilus edulis, Mytilus galloprovincialis and Mytilus trossulus. The latter one is also known as the Baltic mussel and its commercial importance is far less than the two other species27. The blue mussel, M. edulis and the , M. galloprovinciales are the most popular mussels in European kitchens. The former one is mainly distributed among northern waters while the latter one can be found around the Mediterranean Sea, the French and Spanish coasts. Due to their similar phenotype and capability to hybridise in areas where they co-occur, the data presented here may not uniformly refer to one specific species.3 Within the European borders, France, followed by The Netherlands and the UK respectively made up the largest share in the production of the blue mussel in 2010 (Figure II-1).

140

120

Denmark 100 France 80 Germany 60 Ireland

40 Netherlands thousandoftonnes 20 Norway

0 Sweden

United Kingdom

2001 1950 1953 1956 1959 1962 1965 1968 1971 1974 1977 1980 1983 1986 1989 1992 1995 1998 2004 2007 2010 Year

Figure II-1 Trend in production of the blue mussel in Europe.3

LITERATURE STUDY 3

Data from the Food and Agriculture Organisation (FAO, 20103) illustrate the rising importance of aquaculture in bivalve production. When it comes to capture production of the blue mussel, one can see a gradual decline over the past two decades (Figure II-2).

450 400

350 300 250

200 Fisheries 150 Aquaculture in thousand in tonnes 100 50

0

1993 1992 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Year

Figure II-2 Evolution of the production of the blue mussel in the world, by fisheries and aquaculture.3 The most popular mussel farming method in the Netherlands is bottom culture. Young mussel seed (2 cm) is fished from the mussel banks in the Wadden Sea and transferred to designated plots in the Wadden Sea and Eastern Scheldt, where growing conditions are optimal. When they reach the commercial size, the animals are harvested by dredging.28 In France, the Bouchot cultivation is the most commonly applied method: larvae are collected from nature on coconut ropes where they settle. After doubling, seed attached to ropes are then wrapped around stakes. The stakes are driven vertically into the ground and a net prevents the mussels from falling.2 Rope culture from floating devices is also an option but this is usually done for M. galloprovincialis, in the North-west of Spain and the Mediterranean region.1

LITERATURE STUDY 4

II. The host: Blue mussel (Mytilus edulis)

A. Phylum: Class: Bivalivia Subclass: Order: Mytiloidea Superfamily: Mytiloidea Family: Genus: Mytilus Species: edulis

Figure II-3 Taxonomy of Mytilus edulis (Linnaeus, 1758)29. The blue mussel Mytilus edulis, belongs to the subclass of Pteriomorphia, characterised by lamellibranch gills (Figure II-3). These bivalves do not have real teeth on their valves, but the inside of the shell is characterised by a typical nacre layer. The two valves are identical, usually elongated and narrowing towards the frontal region. The anterior adductor mussel is still present, but small. Finally, the members of this family have a mantle which forms a closed exhalent siphon 30.

B. Biology and habitat Blue mussels are active filter feeders. From the early larval stage onwards, cilia are used to remove particles from suspension. Larvae are limited by the size of the food particles because their oesophagus is just about 10 µm wide.31 Adult animals on the other hand are known to retain particles within a size range of 7-35µm, however it has been suggested that they can also control their own particle retention mechanisms to a certain level.32 These food particles mainly comprise unicellular algae and organic material. Also bacteria have been found to make out a significant part of the food, since they serve as a substantial source for the carbon and nitrogen requirements.33,34 Spawning in nature usually happens when the feed load in the water is on an increase, in order the avoid starvation of the newly born larvae.35 Nevertheless, the young larvae have some internal energy back-up, which allows them to survive up to 10 days (at 16°C) without food.36

LITERATURE STUDY 5

The blue mussel is distributed along the boreo-temperate region, in the North-Pacific, North- and Mid-Atlantic up to the Arctic oceans.37

The adult animals can be found in a wide variety of ecological niches, ranging from littoral to shallow sub-littoral and from polyhaline to mesohaline estuarine environments. This broad range of niches resulted in a far-going physiological adaptation. The temperature tolerance therefore mainly depends on the length of the acclimatisation and adaptation period. A lab test estimated an upper lethal temperature of 27-29 °C.38 Low water temperatures are generally well tolerated by adults and some populations in the Labrador Sea have even been known to survive being frozen for 8 months.37 Larvae require temperatures of at least 5°C and growth will progressively increase up to a temperature of 16°C.39 Salinity is also an important parameter, their tolerance towards salinity changes mainly depends on the genotype, interaction with temperature.40

C. Reproduction and life-cycle The blue mussel, is a dioecious species, in which males and females can be distinguished by a slight difference in the colour of the ripe gonads.41 Adult animals are in general ready to spawn from one year onwards, when the reproductive season is there. Under natural conditions, the preparation to this spawning happens during winter time when food availability is limited. The necessary energy for this previtellogenesis, the formation of oogonia, comes from their glycogen reserves.42 The actual vitellogenesis, when oocytes are formed, occurs over a shorter period and involves the storage of lipids in the egg yolk of the female’s gametes. There is not just one trigger responsible for the onset of the reproductive cycle. It is rather a combination of internal factors (the nutrient availability and hormone presence) and external factors (water temperature, salinity, duration of exposure to air and food availability).35,37 Especially phytoplankton availability is very important.43 At the moment of spawning, eggs and sperm are released from their follicles and pass through the gonoduct towards the genital papilla and are subsequently released through the exhalent siphon into the water. 44 An accurate estimation by Thompson (1979) 45, based on three North-American populations, reveals that females can produce up to 107 eggs g-1 tissue dry weight, while males are able to release a corresponding 1.1x1011 sperm cells g-1 tissue dry weight. The fertilised eggs have a diameter of 68-70 µm and will undergo meiotic divisions during which two polar bodies are released. The ciliated embryo will pass through the blastula

LITERATURE STUDY 6 and gastrula stage within 24 hours and develops into the first motile planktonic stadium: the trochophore (48 hours after fertilisation) (Figure II-4).

Figure II-4 General drawing of the development of bivalve embryos from the early trochophore (A) to the fully shelled D-larva (D). The ciliated swimming feeding organ (velum) can be seen in B and early shell valve formation in C. from Helm et al. (2004)41 The duration of the complete larval stage (until settlement) varies between 15 and 35 days, and depends on the population and environmental factors.

A trochophora larva is not yet capable to eat on its own and will rely on its internal yolk material for its energy requirements. The subsequent veliger stages do have a feeding apparatus formed by a circular velum that is foreseen of cilia. The additional role of this velum is keeping the larvae suspended. The first, Prodissoconch I stage, is characterised by a straight-hinge D-shape of a thin transparent shell. When the larvae further develops and clear umbones are visible, biologists will speak of Prodissoconch II-stage veliger larvae. During the next two stages, eyespots and foot will start to develop in the respective eyed-and pediveliger larvae. A fully grown pediveliger will look for a good substrate to settle down37. Once a suitable spot has been found, it will metamorphose into a spat (Figure II-5).

LITERATURE STUDY 7

Figure II-5 Spat attached to green sea weed (left). Juvenile (gaping) mussel (right).

When they reach a size of 1.5 mm shell length they will detach themselves and become planktonic again. Currents will transport them to more densely populated beds of adult mussels, where they will use their byssus threads for the final settlement.37

D. Anatomy and physiology of adult mussels In this section a general description is given concerning several aspects of the anatomy and physiology of adult mussels relevant for this thesis, and a more detailed description on the current knowledge of their immune defence system. The mussel’s anatomy is visualised in Figure II-6.

Figure II-6 General anatomy of the blue mussel, Mytilus edulis.46

LITERATURE STUDY 8

1) Feeding and digestive system

The gills will filter out the food particles and guide them towards to mouth region (Figure II-7).

Figure II-7 Schematic drawing of the position of the organs in bivalves. (from Invertebrates, Second edition, Figure 20.8 (Part6) © 2003 Sinauer Associates, Inc. Labial palps surrounding the mouth select food particles and are able to reject unwanted particles, known as pseudofaeces. The oesophagus is short and widens into the visceral mass which lines the anterior part of the stomach (Figure II-8).

LITERATURE STUDY 9

Figure II-8 Schematic drawing of the bivalve digestive system. (from Invertebrates, Second edition, Figure 20.33 (Part1) © 2003 Sinauer Associates, Inc. The last one is completely surrounded by a dark digestive gland, sometimes referred to as the liver. In the anterior part of the stomach a chitinuous gastric shield can be found. A gelatinous rod, the crystalline style, rotates against this wall and grinds the food particles. Doing so, it releases enzymes which facilitate the digestion process. A complex of ciliated ridges and grooves on the stomach will separate digestible organics from indigestible inorganics. The actual digestion occurs in the digestive cecum. The indigestible particles will exit the posterior site of the stomach via the descending intestine, followed by the ascending intestine and the rectum. Faeces enter the mantle cavity via the anus which is located near the posterior adductor muscle. 30

2) Muscles Mussels are dimyarian species consisting of two adductor muscles (Figure II-6). Their specific condition is referred to as heteromyarian since the anterior adductor muscle is much more reduced than the posterior muscle. The posterior muscle closes the shells and has thus an antagonistic effect compared to the hinge ligament and resilium.41 The adductor muscles are composed of striated muscle fibres for rapid contraction and smooth muscle fibres for tonic contraction. 30 These two muscles, visible through gaping, hold particularly large tissue sinuses.47

LITERATURE STUDY 10

3) Cardiovascular system are characterised a by a typical open cardiovascular system (Figure II-9). The blood which is oxygenated in the gills will flow towards the paired atria and subsequently to a single ventricle. This heart is located within a pericardial cavity, on the posterior side of the hinge. In the more evolved bivalves an anterior and posterior aorta exits the ventricle, but in Mytilus spp. only the anterior ventricle is present.30 From there the hemolymph flows into the hemocoel and diffuses through a series of tissue sinuses.47

Figure II-9 General drawing of the bivalve uro-genital and cirulatory system. (from Invertebrates, Second edition, Figure 20.34 (Part1) © 2003 Sinauer Associates, Inc.)

LITERATURE STUDY 11

4) Nervous system The nervous system in bivalves is symmetric (Figure II-10).

Figure II-10 Schematic drawing of the bivalve nerve system. (from Invertebrates, Second edition, Figure 20.41 (Part1) © 2003 Sinauer Associates, Inc. Near the oesophagus, the cerebropleural ganglion is located, formed due to the fusion of the pleural ganglion on the one hand and the cerebral ganglion on the other hand. Their function is to control the anterior adductor muscle, the anterior part of the mantle, the labial palps and the mouth. Two nerve strings run from each ganglion, one that directs towards the pedal ganglion another that goes to the visceral ganglion. Whereas pedal ganglia control the foot, the visceral ganglia control the posterior adductor muscle, the gills and the posterior edge of the mantle, including the siphons.30

LITERATURE STUDY 12

5) Immune defence system As it is true for all invertebrates, bivalves are characterised by an innate immune system.48 This type of system, also known as the non-specific immune system, meaning that the recognition and response towards a pathogen occurs in a systematic way. Unlike with an adaptive immune system the organism cannot benefit from a long-lasting immunity. The immune response of bivalves can be subdivided into two (overlapping) mechanisms, a cellular response and a humeral response.49

The role of the cellular response is taken up by blood cells referred to as hemocytes, present in the hemolymph of bivalves23. A simple division into granulocytes on the one hand and agranulocytes (hyalinocytes) on the other hand is overall accepted.51,52 The first group represents the most abundant type of hemocytes, they contain hydrolytic and oxidative enzymes and are more active in phagocytosis than hyalinocytes. 53 The latter ones are smaller and more morphologically heterogeneous. A generally accepted detailed classification of bivalve hemocytes is still lacking because of the use of different description criteria e.g. morphology, cytochemistry, function, etc. 50

Phagocytosis is the process where hemocytes engulf foreign compounds e.g. bacteria. Before the actual phagocytosis can take place the hemocytes need to come in the vicinity of their target. This can be enhanced by two motile responses, a directional movement (chemotaxis) or an indirectional, random movement (chemokinesis) of the blood cells (Figure II-11).

Figure II-11 Basic difference between chemotaxis and chemokinesis. The trigger for this movement is a (set of) chemical(s) to which the hemocytes are sensitive. In M. edulis for example, the presence of lipopolysacharides from E. coli and Serratia marcescens

LITERATURE STUDY 13 resulted in the directional movement of hemocytes (chemotaxis) in vitro. N-formyl-methionyl- leucyl-phenylal- anine (N-FMLP) on the other hand just stimulated random movement of the bloodcells54. Nevertheless, both reactions might result in a higher probability of an encounter between hemocyte and invader in vivo.55 When the hemocyte approaches the bacterium, they have to recognise the non-self compound. In opsonophagocytosis this will be mediated by molecules called opsonins, which will bind the antigen and will make recognition of this antigen by the phagocytic hemocyte possible. A widely recognised group of molecules that can function as opsoninising factors in bivalves are lectins.56– 59 This type of phagocytosis has been demonstrated in Mytilus edulis58. Beside opsonisation, lectins can also have a agglutinative action towards the bacterium, with phagocytosis as a secondary consequence55. The contrary of opsonic phagocytosis is non-opsonic phagocytosis and in this process, receptors on the cell wall of blood cells will directly bind to macromolecules on the cell wall of the bacterium.55 At the moment a bacterium has been recognised as being an invader, it will be invaginated and internalised (endocytosis) by the hemocyte and a primary phagosome will be formed.60 The latter one will then fuse with lysosomal granules and form the secondary phagosome. At this moment, the lysosomal enzymes, which are mainly acid hydrolases61 (e.g. acid phosphatise, esterase), start digest the phagosome content. Phenoloxidases is another group of enzymes which is now being recognised to play an important role in bivalve immunity.62 Toxic oxygen intermediates and even antimicrobial peptides can also at this moment be important in the degradation of the invader.63 Studies performed to assess the immune response of larvae or adult animals, will use these molecules, or the mRNA of immune system related genes as indicators of a respons.64–66

The hemolymph of bivalves also contains humoral factors, molecules that can have a direct cytotoxic effect on bacteria 67. It should be kept in mind that some of these components are also present inside the haemocytes, and thus play a role in intracellular digestion and cellular defense as well. Research on humoral factors has increased in the last decade and Table II-1 summarises some important studies for each corresponding group.

LITERATURE STUDY 14

Group Function Reference Lysozyme Digestion and destruction of 52,68–79 bacterial cell wall

Lectins opsonising factors, 67,79–81 agglutination

Antimicrobial peptides Destruction of cell wall and 63–66,82–87 cytosolic components

Table II-1 Most important humoral factors found in Mytilus-species. A first group of antibacterial molecules that can be found within the hemolymph is lysozyme. It is an enzyme that is widely distributed among different classes and is characterised by its capacity to hydrolyse β-1,4-linked glycoside bonds of the bacterial cell wall peptidoglycans.88 Lysozyme can be found associated with the crystalline style sac, soft tissue, hemocytes and hemolymphe of mussels. 68 It is temperature dependent as well as pH-dependent and holds an additional chitinase activity.89 Secondly there are the lectins, which were already mentioned before in the part on phagocytosis where they can act as opsonising factors. They are sugar binding proteins that specifically and reversibly bind to glycans on living cells.90 Beside their known presence in haemocytes and haemolymph they have recently been traced in the mucus of the gills and labial palps.91 Their mode of action probably involves the immobilisation of bacteria by agglutination. However it is not sure if this is a final step, or the first step of the phagocytic process (opsonizing step).52 Another, and probably one of the most important groups of humoral factors are the antimicrobial peptides. They are usually characterised by their small size, heat stability and broad range of antimicrobial activity.92 The most commonly investigated AMP’s are; mytilins, myticines, mytimycins and defensins. However they can be secreted by the hemocytes into the blood, their activity during phagocytosis has also been recognised.83 The mechanism of action towards bacterial cells of the free AMPs involves several consequent steps. Electrostatic attraction to the cell membrane results in stepwise accumulation of the peptides on the membrane. When the threshold has been reached the peptides will deform the membrane and insert into it. Subsequently, they may form complexes and be finally translocated to the cytoplasmic face of the membrane where they will act on cytosolic components.82 Since they have no cytotoxic effect on

LITERATURE STUDY 15

the host, these molecules are of great interest for the use as immunostimulants, in order to prevent diseases in aquatic organisms.92

III. Pathogens & associated diseases One of methods used to define a “pathogen” are Koch’s postulates93,94;

1. The causative agent has to be present in large quantities in the animal.

2. It should be possible to extract this agent and culture it again.

3. The larvae that are inoculated with this agent, should show the same disease symptoms.

4. The causative agent should be isolated from the experimental larvae and should be exactly the same as the ones, first detected in the original population of diseased animals.

Moreover it has to be kept in mind that the susceptibility of the host towards the pathogen might be dependent on the life stage.52,61,95 Before going more into detail on the aspect of diseases associated with larvae and adults respectively, a brief overview is given of the bivalve pathogens that are known nowadays.

A. Diversity of pathogens Pathogens causing bivalve diseases can be of a highly diverse origin; it can be viruses, bacteria, protozoa or fungi. 96–99 Within the scope of this thesis only the bacterial causative agents will be discussed. Paillard et al. (2004) and Beaz-Hidalgo et al. (2010) gave an overview of pathogenic bacteria for bivalves.24,94 The most important disease causing agents reported are: Chlamidia, Vibrio, Alteromonas, Aeromonas, Pseudomonas, Cytophaga, Nocardia, Mycoplasme and Ricketsia. Regardless the high diversity in the causative agents at first sight , about 50% of the reports of diseases are caused by Vibrio spp.94. Pathogenic Vibrios of bivalves are; V. tubiashii95,100–103; V. alginolyticus100,104–107; V. anguillarum100,104,108–113; V. splendidus93,103,114,115, V. splendidus biovar II107,116 and V. splendidus-like117; V.pectinicida118–120; V. tapesi121; V. tapetis122–126; V. neptunius127; V. cholerae103; V. aesturianus128–130 and V. aesturianus subsp. francensis131 and some unidentified Vibrio spp.95,102,117,127,132–141

LITERATURE STUDY 16

Except for V. alginolyticus and V. anguillarum, most of these bacteria have not been reported to be pathogenic for M. edulis yet. 142,143

B. Larval diseases Bacillary necrosis is the most common disease among bivalve larvae. Guillard et al. (1959)137 first isolated the pathogenic agents from hard clam larvae and Tubiash et al. (1965;1970)95,104 further identified these bacteria. This disease is most likely to be associated with V. alginolyticus, V. tubiashii, V. anguillarum102,104,109,V. splendidus114 and V. pectinicida115

First symptoms of bacillary necrosis can show early as 4-5 hours after exposure.104 In hatcheries, typical signs are a reduced motility, swarming of the bacteria around the larvae, originating from discrete foci on the margins of the larvae, and quiescent larvae lying on the bottom of the well or tank. This sedimentation of immotile larva is a well-known phenomenon and is named spotting.110 The immotile larvae is usually characterised by an extended foot and velum, and the latter one shows signs of decilliation of the epithelial cells.135 In a more progressive stage of infection, histological sections will reveal cellular destruction to a high degree95.

Elston and Leibovitz (1980)135 divided this disease in three patterns, according to the stage at which Vibrio spp. will be the most virulent. In pathogenesis I, larvae of all stages will be affected and most obvious symptoms are the colonisation of the mantle and invasion of the visceral cavity, together with a reduced mobility. The second pattern, pathogenesis II only affects early veliger larvae, causes velum disruption and extension, and has an effect on the swimming capacity. The larvae will remain active until bacteria invade organs of the digestive tract, signs of visceral atrophy can be seen. The parthenogenesis III will only affect the late larval stage and is characterised by immobile larvae. Additionally, there can be seen a progressive and extensive visceral atrophy and lesions in the organs of the digestive tract24. Signs of bacillary necrosis are mainly reported in hatcheries of oysters, clams and scallops.100,104,105Yet, none of these symptoms have been described for the larvae of Mytilus edulis, though similar symptoms have been observed in M. galloprovincialis.106

C. Juvenile and adult diseases Bacterial diseases which are associated with juvenile bivalves are; Brown Ring Disease144, Nocardiosis140, Juvenile Oyster Disease and the summer mortalities of the Pacific oysters.93,116

LITERATURE STUDY 17

Vibrio’s which have been reported to infect adult bivalves are; V. tapeti125,126s, V. splendidus93,116 and V. aestuarianus129,130. Whereas some of these bacteria caused instant mortality93,116 when they manifested in bivalve cultivation plants, others showed clear disease symptoms during experimental infection e.g. weakening of the adductor muscle.117 Less obvious symptoms seen during experimental infections were reported as well e.g. reduction of phagocytotic capacity of the haemocytes.124,126,128. All the diseases mentioned above were reported for commercially interesting species like clams, scallops and oysters. Just a few descriptions of Vibrio-interactions were associated with Mytilus- species, mainly with Mytilus galloprovincialis as host, but it only concerned in vitro effects (e.g. increased AMP expression, ROS production etc.). 66,142,145 Based on the literature review, a selection was mad of potentially pathogenic bacteria (Table II-2).

Species BCCM-Code Reference Host Stage 145

Listonella anguillarum LMG 04437 M. galloprovincialis adults 146

Vibrio alginolyticus LMG 04409 M. galloprovincialis larvae Vibrio harveyi BB120 147 Vibrio proteolyticus LMG 10942 148 O. edulis larvae Table II-2 Selected Vibrio strains from literature.

D. The use of rifampicin To minimise the presence and influence of unknown and unwanted bacteria in the development of a challenge test, all tested animals in the setup of this thesis were treated with the antibiotic rifampicin and rifampicin resistant strains were used for the in vivo experiments. An additional advantage is that the use of rifampicin makes it possible to trace back the bacteria of challenge, by plating host tissue/tank water on a selective medium (e.g. MA+RIF). Rifampicin (Figure II-12) is an bacteriostatic antibiotic derived from the bacterium Amycolatopsis rifamycinica, and belongs to the rifamicin group of antibiotics149–151.

LITERATURE STUDY 18

Figure II-12 Chemical structure of rifampicin. It will inhibit the transcription process by inactivating the RNA polymerase of bacteria due to the formation of a stable enzyme-drug complex. More precise, it is the β-subunit of RNA-polymerase which is the target of the antibiotic. In other words, this drug will prevent the initiation of the chain formation and by doing so it avert the synthesis of messenger RNA.152,153 Rifampicin is mainly effective against prokaryotic RNA-polymerase, mostly towards gram-positive bacteria, and in a lower extent towards gram-negative bacteria.151,154 It also has no effect on RNA- polymerase of eukaryotes because of its different structure. 150,155 Resistance towards this drug originates from a mutation in the rpoB gene, the sequence that codes for the β-subunit enzyme of RNA-polymerase.156 Many research has been done on rifampicin resistance of Mycobacterium tuberculosis157–160 . Regarding this bacterium, mutations use to occur within codons in the so called “hot spot” region and different mutations can result in a different degree of resistance.161 A point mutation like this will result in the insertion of another amino acid in the β-subunit of RNA polymerase. Since the rifampicin-βsubunit complex gets its strength from a bound between naphtoquinone on the on the one hand and aromatic aminoacids on the other hand, replacement of these aromatic amino acids by non-aromatic amino acids, subsequently results in a significant decrease in strength of the complex.162

LITERATURE STUDY 19

IV. Environment Finally, the role of the environment cannot be underestimated. Various factors, ranging from salinity, temperature, CO2 levels, pH, dissolved oxygen concentration (DOC), physical disturbance over anthropogenic pollutants to feed availability will influence the host susceptibility on the one hand, and the pathogens growth and virulence characteristics on the other hand.163 Fluctuations in optimal and suboptimal environmental conditions might lead to an increased susceptibility to disease.164–166 It is not possible in the time frame of this thesis to discuss all these different environmental aspects and their possible influence on disease susceptibility in bivalves. Therefore, it was decided to limit ourselves to study the effect of stress regarding the host, in terms of physical disturbance. 162 The role of the environment for the pathogen will be evaluated by increasing the nutrient load of the water. Since the potential pathogens for M. edulis are heterotrophic, the effect of a high organic load of the water might be an interesting factor to evaluate. It is expected to have a beneficial effect on the growth and virulence of the bacteria.164

V. Critical discussion of important related research Because of the high dependency on natural spat fall, hatcheries for M. edulis larvae are rather scarce compared to those of high valuable bivalves like oysters, scallops and clams. As a consequence, hatchery problems are also less common the subject of research. Beaz-Hidalgo et al (2010)24 recently summarised the findings of experimental infection tests with Vibrios on bivalve larvae and adults, and gave a good overview of the current knowledge. By addressing the basic questions below I will try get an overview of the important aspects of larval and adult bioassays will be listed.

1. How are bacteria collected, purified and stored; 2. How are larvae/adults inoculated and how are sterile conditions assured; 3. How are the bacteria administered; 4. What parameters can be monitored during challenge?

A. Larvae In case the bacterial strain of interest did not originate from a databank, it was isolated from diseased animals. Tissue parts or whole animals can be grounded and the manifesting bacteria

LITERATURE STUDY 20 should be isolated in a aseptic manner.105,127 In order to possess pure cultures it is advisable grow and pick-up single colonies from a plate and repeat this step for one or two times more.127 If long- terms storage is necessary, the samples can be stored in the freezer at -80°C. Glycerol is usually added, but the exact percentage varies from 10-25%.107,140

The larvae can be challenged from the earliest stage of development, the trochophora larva, or at a later stage.106,119,127 Most recent studies used a high variety of types of tanks and culture wells to inoculate their larvae. Some use rather large experimental units of 1L106, while others use small cell-culture microplates127 or 24-well multidish trays119. The larval density in every experimental unit is generally kept at a rather low level ranging from 10-50 larvae ml-1.105,119,127,140 In the studies I read, the scientist took certain measures to minimise and control the degree of contamination. This mainly consists of autoclaving the equipment and filtering (0.45 or 0.22 µm) and autoclaving of the sea water they used for the inoculation of the larvae. 107,127,140 Anguiano- Béltran et al. (2004) went a bit further by washing recently fertilised eggs with 0.45 µm filtered sea water and incubating them immediately in the experimental units filled with SSW. Plating of water samples did not reveal the presence of Vibrio-like bacteria, but whether these measures are enough can be discussed.

Bioassays have to be done inevitably in ways of in-bath treatment, considering the small size of the larvae. The bacteria strains of interest are often cultured in a certain type of broth. Preliminary experiments are advisable to rule out toxic effects of the broth. If this is the case, it can be decided to wash the cells in phosphate-buffered saline by centrifugation.105,107,119Alternatively, the strains can be grown on plates overnight, and colonies can be swapped off and be suspended in SSW.127 Although these are the most popular methods, not all experiments are done with whole cells. Some scientist decided to challenge the larvae with the culture supernatants or extracellular products.103,107,168 The cell concentration can be measured in several ways; OD-measurement, the McFarland method or via plating and enumeration.106,119,127 The duration of the entire experiment can vary from 24 hours to several days.119,127Some authors mentioned that they fed the larvae during the experiment. Although this extra procedure increases the risk of contamination, they claim that the algae mixture did not contain TCBS-positive bacteria.105

LITERATURE STUDY 21

Finally, a good way of quantifying the effect of the bacteria towards the larvae is essential. Some researchers restrict themselves to calculating the percentage survival105. Others include parameters like; swimming/non-swimming, bacterial swarming around the larvae127, and presence or absence of symptoms of bacillary necrosis. For the latter parameter, tissue slides can be made in case of larger spat (6 mm), which can be subjected to histopathological analysis.107 If different concentrations of bacteria were used, the LD50-value can be calculated as well.

B. Juveniles and adults The isolation, storage and preparation of the challenging bacteria for juvenile and adult bioassays are identical as for the larval test.

Many practical aspects of adult challenge test however are quite different. If the test animals are collected from the wild, they can be acclimatised for 2-3 days to the laboratory conditions.169 Also the experimental units for juveniles and adults do differ significantly with those of larvae. The juvenile or adult animals are usually kept in larger aquaria filled with aerated sea water (filtered and/or artificial).107,117 When the animals are fed and the experiment is performed over a long period, the water has to be changed regularly, or a flow-through/recirculating system has to be installed.93 It is obvious that these circumstances increase the risk of (cross-) contamination, and good hygienic practices are therefore essential.

Where for the larval experiments in-bath treatment with the pathogen is the only option, there are more possibilities when it comes to adults. Exposing the animals to sea water with a high load of the bacteria of interest is also done, but many scientists prefer to inject the bacterial solution directly into the animal. This injection takes place in the posterior adductor muscle of the bivalve.93,130 Another type of challenge involves the in vitro follow-up of the effect of the pathogen. Layers of the desired cell type, e.g. hemocytes, can be exposed to different bacterial strains and the behaviour can be monitored over time.143

Monitoring the effect of the challenging pathogen is in the first place done by recording the survival percentage. Due to the larger size of the adults compared to the larvae, it is more common to perform additional histological analyses, or to withdraw hemocytes in order to assess the viability effect of the bacteria on these cell types.5 Many complex parameters are recently getting more attention from scientists to monitor the effect of a pathogen towards adult bivalves,

LITERATURE STUDY 22 e.g. reactive oxygen production, nitrogen oxygen production, AMP expression etc.66,145, but discussing every technique is out of the scope of this literature review.

Challenge experiments with adult blue mussels are few.McHenery and Birkbeck (1986)170 first observed inhibition of filtration of Mytilus sp. in the presence of high concentrations of Vibrios. In a later study142 mussel hemocytes monolayers were created and both intact V. alginolyticus- cells and culture supernatant was added to observe their effect on the hemocytes. They saw that for the virulent strain (NCMB1339) the intact cells did induce more significant effects than the supernatant, but when the non-virulent was used, the opposite effect was seen. Lane and Birkbeck143(1999) calls the ability to induce hemocyte rounding essential for a bacteria to be toxic towards these cells. The same V. alginolyticus (NCMB1339) was tested in their experiments, together with a V. anguillarum strain (A7) and showed similar results. In a later study they reported even more strains which were toxic towards M. edulis hemocytes.171 On first sight, the above mentioned bacterial strain sounds interesting, nevertheless, it should be noted that these results were obtained with filtered hemocytes and that tests with complete hemolymph did result in very low survival of the challenged bacteria.142

LITERATURE STUDY 23

PART III. MATERIALS AND METHODS

A subdivision is made into three parts

- Standard materials and methods: All techniques used on a general basis during the practical work of this thesis are grouped in this part to avoid needless repetition. - Development of a challenge test: The different experiments performed in the process of developing a challenge test are explained chronologically. The method used to analyse the diversity among the isolated strains is explained as well. - Development of a molecular toolbox to study the expression of immune related genes : the material & methods used for the selection of relevant immune related genes, followed by the design and testing of primers for immune gene expression analysis is explained here

I. Standard materials and methods

A. Vibrio strains

In Table VIII-1 (Annex 1) a summary is given of all the bacterial strains that were used during this thesis. Some of the strains were already present in the lab. Others were ordered from the Belgian Co-ordinated Collections of Micro-organisms/Laboratory of Microbiology (BCCMTM/LMG), based on previous scientific reports. 172 Finally, we also isolated bacteria from dead and moribund animals during the experiments.

B. Culture media

1) Marine agar (MA)

55.1 g of marine agar (Difco Marine Agar TM) was suspended in 1 litre of distilled water. The solution was autoclaved for 20 minutes at 121°C. Plates were subsequently poured in the laminar flow and stored in plastic bags at room temperature for further use.

2) Thiosulphate-citrate-bile salts-sucrose (TCBS)

88.1 g of TCBS (Biokar Diagnostics) was suspended in 1 litre of distilled water. The solution was mixed with a magnetic stirrer, while it was heated up to boiling point. Plates were

MATERIALS AND METHODS 24 subsequently poured in the laminar flow and stored in plastic bags at room temperature for further use.

3) Luria-Bertani (LB) broth marine

10 g of bacto tryptone (Biokar Diagnostics), 5 g of yeast extract (Fisher Scientific) and 35 g of Instant Ocean was dissolved in 1 litre of distilled water. The solution was then autoclaved for 20 minutes at 121°C. The LB broth was stored at room temperature for further use.

C. Growing of bacteria

Bacteria that had to be grown on liquid medium were always inoculated in a falcon tube containing 5 ml LB marine. The falcon tubes were then placed on a shaker (G24 Env. Inc. Shaker, Edison N.J.) and incubated overnight at 30°C. In the case a plate culture was required, 50-100 µl of broth cultured bacteria was plated on MA or TCBS using a triangular spatula. Alternatively, bacteria were also streaked with a sterile needle. Then the plates were incubated at 30°C in the oven (LedTechno). Single-strain, dense bacterial cultures (107-108) were transferred to 40% glycerol (Vel n.v.) and stored at -80°C for further usage.173

D. Rifampicin resistance

In a first step the bacterial strain of interest was grown overnight at 30°C on LB marine. The desired bacterial load was 108-109 CFU ml-1. The next morning, 500µl of this bacterial suspension was then transferred to 5 ml of LB broth, into which rifampicin (SIGMA) was added, at a concentration of 50 mg L-1. The rifampicin was diluted in 0.1 N HCl and filtered (0.2 µm), prior to adding it to the LB marine broth. It could take a few days before the medium showed signs of growth, because the initial number of rifampicin resistant bacteria, that are thus capable to grow in this environment, is rather low.

E. Api ZYM (Biomérieux)

In a first step the bacterial strain of interest had to be grown overnight on LB broth. The next day the api ZYM test was prepared. Prior to adding the strips to the lids, 5 mL of distilled water was distributed equally over the honey-combed wells in the bottom of the lid, in order to create a humid atmosphere in the box. Then the test strip was added to the box, and every cupule was MATERIALS AND METHODS 25 filled with 65 µl of bacterial suspension. When all the bacteria were inoculated on a separated strip, these strips were incubated in the oven at 37 °C. After 4 hours the strips were taken out of the oven. One drop of ZYM A and ZYM B was added to each cupule. About 5 minutes later the result could be read. The intensity of the reaction was scaled from 0 to 5 (0: no reaction, 5: positive reaction) and resulted in a specific enzymatic profile for the tested strain.174

F. Test animals

1) Adults

The blue mussels used for these experiments were initially harvested from the Dutch part of the North Sea. All but the first batches arrived unprocessed at the lab, originating from the company Roem van Yerseke. They were transported to the lab under cooled dry conditions . If the mussels had not passed through the cleaning machine, they had to be cleaned at the lab. Barnacles and other fouling organisms were removed and dirt was washed away. When the animals had been processed already, thorough rinsing was sufficient. Subsequently, all animals were acclimatised (for how long) in a rectangular tank (79 cm x 59 cm x 30 cm) filled with 0.45 µm filtered sea water and aeration

2) Larvae

The Mytilus edulis larvae used in the first challenge experiment were produced in the ARC lab. The broodstock was brought from Roem van Yerseke and the animals were ready to spawn. Males and females were stimulated to release their gametes according the thermal cycling method41. Spawning animals were separated and their gametes were mixed in a beaker. The ratio of sperm versus egg cells was kept around 10:1. After fertilisation the development was monitored regularly. When the morella stage was reached in the majority of the embryos, the embryo solution was brought on a 30 µm sieve and the remaining sperm was washed away with 0.45 µm filtered sea water. After this washing step, the embryos were transferredto a tank (l?) with filtered sea water (21-22°C) treated with rifampicine (20mgL-1) where they continued to develop in D-larvae after 48 hr.

MATERIALS AND METHODS 26

G. Anaesthesia procedure

-1 175,176 The mussels were transferred to a tank, containing MgCl2 (Acros Organics) (28g L ) . In order to reach a salinity of 35 psu, 3 litres of sea water had to be mixed with 5 litres of fresh water. Aeration stones were provided and the water parameters, temperature, pH and salinity, were measured to assure similar conditions in both treatments.

H. Disinfection

All equipment needed, was thoroughly cleaned, disinfected with 2% CID 2000® and rinsed before and after usage. Additionally, the floor was cleaned & disinfected with 1% ViroCid® and the surfaces with Detol. In the laminar flow, the table and the materials were disinfected with Disinfectol®.

MATERIALS AND METHODS 27

II. Development of a challenge test for Mytilus edulis

A. Larval experiment

The first experiment performed in the scope of this thesis had the purpose;

- to evaluate the material and methods that are used

- to screen 5 selected bacterial strains on their potential virulent effect on mussel larvae (annex 1, Table VIII-1)

- to decide whether the addition of algae or trypton influences the larval survival/mortality rate

Bacteria

Five bacterial strains, previously made rifampicin-resistant were grown one day prior to the actual challenge on LB marine at 22 °C. The next day, the bacteria were harvested and re- inoculated in order to assure a challenge with live bacteria (only 7-8 hour old).

The algae

Algae of the genera Tetraselmis and Isocrysis were grown about one week in advance in autoclaved seawater, supplied with Walne medium and vitamins. They were grown in a conditioned room under continuous illumination, and hygienic although non-sterile conditions.

The challenge

The 2-day old larvae which had developed in seawater treated with rifampicin (20 mgL-1)), were harvested upon a 60 µm sieve and washed thoroughly with 0.45 µm filtered sea water of 22°C. After re-suspending the larvae, the concentration was checked and corrected in order to obtain a final concentration of 250 larvae ml-1. The concentration of the algae in the Erlenmeyer was assessed by counting 4 sub-samples in a Bürcker counting chamber. For the inoculation of the larvae, 24-well plates were used. Rifampicin was added to all the wells, while the algae and trypton (Biokar, Diagnostics) were only added in the respective treatment groups (Figure III-2). The Vibrios were administered at 105 cells ml-1. The experiment was performed in quadruplicate. Three identical plates were made for each treatment, because at each sampling moment one plate had to be sacrificed for counting. MATERIALS AND METHODS 28

After inoculation of all wells, the plates were brought to a conditioned room at 21°C. The next day, health and survival was monitored visually. 37 hours later, the first series of larvae was stained with lugol and counted under a binocular (Euromex, Holland). The next two counts took place, respectively at 61 and 83 hours p.i..

V1 V2 V3 V4 V5 -1 Larvae (250 ml ) + Rifampicin (20mg L-1)

V1 V2 V3 V4 V5 Larvae (250ml-1)

+ Rifampicin (20mg L-1)

+ Algae (25* 103 cells ml-1)

V1 V2 V3 V4 V5 Larvae (250 ml-1)

+ Rifampicin (20mg L-1)

+ Trypton (1mg L-1)

Figure III-2 Schematic presentation of the larval challenge experiment. Three plates per time interval ( 37 hrs, 61 hrs and 83 5 p.i.). V1: LMG 04409RIF, V2: BB120RIF, V3: LMG 11257RIF, V4: LMG 4437RIF, (10 cells/ml) C: Control

MATERIALS AND METHODS 29

B. Adults Regarding the adult test discrimination can be made between:

1) In vivo experiments with bacteria 2) Anaesthesia experiment 3) The effect of an enriched environment and physical stress of the host

1) In vivo tests with bacterial strains

Three different adult in vivo experiments with bacteria were executed:

a) Screening with 7 different strains b) In vivo test with LMG 11229RIF (V. tubiashii) and LMG4437 (V. anguillarum)

c) In vivo test with 2ARIFResistant and 2Awildtype

In a first screening test, different bacteria strains were injected. The selection of these strains was based on findings from literature (Annex 1, Table VIII-1). The screening was performed on 20 animals. Five animals were sacrificed after 24 hours of injection in order to determine the bacterial clearance rate. The remaining 15 animals were kept for long term survival monitoring.

For the second test, 2 of the most promising strains (based on the results of the screening test) together with 2 newly isolated strains were selected (Annex 1, Table VIII-1). At different time intervals (0,5, 1, 2, 4, 8, 16, hours p.i.) hemolymph samples were taken for; 1) determination of the bacterial clearance rate and 2) RNA based immune gene expression research. These tests were executed in triplicate (3 animals/replicate/ time interval). Additionally, 20 animals were kept for survival monitoring.

Bacteria

50 µl of each rifampicin resistant pure strain (stored at -80°C) was incubated overnight in LB marine at 30°C on a shaker and was plated on solid medium, containing rifampicin (50 mg L-1).

The next day, multiple colonies of each strain were picked-up with a sterile plastic needle (BD PlastikpakTM) and suspended in a sterile 2 ml-well, containing 0.2 µm filtered and autoclaved sea

MATERIALS AND METHODS 30 water. The OD550 value of this solution was measured with a spectrophotometer (Gensys 20, ThermoSpectronic). The resulting measurement was then used to further dilute the bacterial solution, in order to reach an end concentration of 1x108 cells ml-1 for each strain.

The challenge

About 2 hours prior to the challenge, the animals were anaesthetised according to the procedure described before. 100 µl of bacterial suspension (108 cells /ml) was injected in the posterior adductor muscle using a 1ml syringe with a 26 G needle. For each bacteria strain another syringe was taken and the animals were injected randomly. After injection the animals were transferred to 36 x 21 x 15 cm rectangular tanks, filled with 0.45 µm filtered sea water (10 animals/ tank in first experiment, 15/ tank later on). The oxygen level was maintained with aeration stones. The water of each tank was renewed on a daily basis with acclimatised water

Hemolymph collection

0.5 ml of hemolymph was taken from the posterior adductor muscle of 3-5 animals per replicate and was pooled into a 1 ml-syringe. Depending on the purpose of the blood sample, it was immediately frozen in liquid nitrogen or plated on TCBS (RIF) or MA (+RIF). For the exact working procedure during the in vivo tests with LMG4437RIF, LMG 11229RIF and 2ARIF, I refer to the tables in annex 2 (Table VIII-2 and Table VIII-3).

2) Anaesthesia experiment All the animals received a rifampicin treatment before the onset of the experiment to avoid unwanted bacterial influence on the mortality-survival rate during this experiment -1 160 mussels were transferred to a tank, containing MgCl2 (28g L ). In order to reach a salinity of 35 (g/L), 3 litres of sea water had to be mixed with 5 litres of fresh water. Another 160 mussel were transferred to another tank and served as a control group. Aeration stones were provided and water parameters, temperature, pH and salinity, were measured in both tanks with to assure similar conditions in both treatments. When one hour had passed, a series of 20 animals was taken from each treatment, which was then subdivided among two rectangular aquaria, functioning as replicates. This procedure was repeated for the next 7 hours in order to test the effect of 0 up to 8 hours of sedation time. The

MATERIALS AND METHODS 31 animals did not receive any aeration during their entire recovery period, due to the lack of sufficient aeration equipment.

3) The effect of an enriched environment and physical stress of the host

In this experiment the effect of physical stress (shaking) and the enrichment of the rearing water by addition of LB, was evaluated without the addition of extra bacteria besides those naturally present in/on the mussels

The rectangular tanks were stocked with 20 animals. The water was changed daily and aeration was provided. Additionally, plastic foil was used to cover the tanks avoiding cross contamination In a first screening test, 16 different treatment combinations were tested (Annex 3,Table VIII-4) without replicates. For the second screening, 5 of the most promising treatments (based on the results of the screening test) were selected and run in triplicate (Annex 3, Table VIII-5).

Rifampicin was dosed at a concentration of 20 mg L-1 and LB marine at a concentration 10ml L-1. The effect of physical disturbance on the mussels was done by shaking the animals in their tank for 30 seconds, prior to adding the water. The anaesthetic was applied at a concentration of 28 -1 grams MgCl2 L for 2 hours. The mortality was recorded every day during the water exchange. If it was high, the hemolymph of a living animal was taken and plated on TCBS. Plates were incubated overnight and a multitude of individual colonies were sampled and pure cultures were grown and finally stored in the freezer. Additionally, a couple of dead animal were crushed with a tissue homogenizer and the sample was frozen at -80 °C in 40% glycerol for future isolation of the bacteria. The water quality was monitored on a regular basis (temperature, oxygen concentration, ammonium and nitrite (JBL, Germany)).

MATERIALS AND METHODS 32

C. Diversity of the isolated strains

The bacterial strains that are isolated during the mortality events will be analysed using an ERIC PCR. This will produce a specific ERIC DNA fingerprint for each strain, and as such give an idea about the diversity among them.

1) DNA extraction For the first bacterial DNA extraction a simple method was applied. 57 bacterial isolates, stored at -80°C were grown overnight in LB marine. The next day, 1 ml of each culture was transferred to a sterile eppendorf tube and centrifuged (Harrier 18/80, Sanyo) for 5 minutes at 13 000 x g. The supernatant was discharged and the remaining pellet containing the bacteria was dissolved in 1ml of PCR water. The solution was then put on a floating carrier and suspended in a bath of water. The water was brought to boiling point and was allowed to boil for 10 minutes. This mixture was then stored overnight at -80°C.

The second bacterial extraction was performed by using a commercial DNA extraction kit (Promega- Wizard® Genomic DNA Purification Kit, A1120), and is explained in more detail in the next section.

2) ERIC PCR

The ERIC sequences are 126 bp long and appear to be restricted to the transcribed regions of the genome.177 When appropriate primers are used, the PCR will generate multiple amplification products, reflecting the distance polymorphism between adjacent DNA repeats.178,179This molecular tool can help us to identify the diversity among the Vibrio strains that were isolated during this thesis. Information about the ERIC primers can be found in Table III-1.

Name Reference Nucleotide sequence Melting temperature (°C) ERIC1 177 ATG-TAA-GCT-CCT-GGG-GAT-TCA-C 65.1°C ERIC2 177 AAG-TAA-GTG-ACT-GGG-GTG-AGC-G 66.4°C Table III-1 ERIC primer sequences and melting temperature according to Eurogentec. The PCR was executed again with MyCycler (Bio-Rad). To optimise the result, two different primers concentrations (2 and 0.5µM) were tested, as well as three different DNA concentrations.

MATERIALS AND METHODS 33

PCR tubes were filled with 25 µl master mix, containing; 2.5 µl of 10x Taq buffer (100mM Tris-

HCl (pH 8.8), 500mM KCl, and 0.8% (v/v) Nonidet P40) (Fermentas); 2.5µl of 25mM MgCl2 (Fermentas); 0.5 µl of 10mM dNTPmix (dATP, dGTP, dCTP and dTTP) (Fermentas); 0.25 µl of Taq polymerase (5U µl-1 (Fermentas)); 5 µl/1.25µl of each primer at 10µM, 1 µl of DNA extract, and 9.25µl/15.25 µl of PCR water (SIGMA).

The amplification reaction consisted of 35 cycles, each cycle programmed for initial denaturation at 90°C for 5 min, followed by 35 cycles of denaturation at 90°C for 30 sec., primer annealing at variable temperatures for 1 min, extension at 72°C for 10 min., and a final delay step of 72°C for 20 min. The different annealing temperatures according this program that were tested during this thesis are; 45°C and 51°C.

3) Gel electrophoresis

Preparation of gel

10 ml of Tris-actate-EDTA (AppliChem, GmbH) stock solution (50x) was diluted 100 times. 50 ml of this solution was mixed with agarose (EuroGentec)(0.5g for first 2 times and 1,0g later on) and heated in a the microwave until the boiling point had been reached. Then, 1µl of GelRed 10.000x (Biotium), was added and the solution was poured into a gel pouring chamber, foreseen of a toothcomb, when the temperature had been dropped under 55°C. The resulting gel was left for 15 minutes to solidify, then put in the electrophoresis apparatus and the buffer solution was added.

Loading of the gel

Small spots of loading buffer (~2µl) were lined up on a sheet of parafilm in a laminar flow. Each drop was mixed with 5µl of PCR-product. Additionally, some drops, free of DNA, were mixed with 4µl of 10x diluted ladder (Fermentas, Mass Ruler SM0313/SM0813). The mixed DNA samples were then loaded into the holes of the gel. When all samples were loaded they were allowed to migrate for about 2 hours at 75V/ 30 min at 100V. Before the samples reached the end of the gel, the machine was turned off and the gel was subsequently transferred to a black glass plate of UV-light (Sungene, Synoptics Group), where the picture was taken.

MATERIALS AND METHODS 34

III. Development of a molecular toolbox to study the expression of immune genes

A. Selection of immune system related genes A set of 9 proteins was initially selected for the experiment, based on a literature study (Table III-2). Due to the lack of sequence data for some of the genes in M. edulis, we ended up with a set of 3 primer pairs each specific for an immune-related gene (lysozyme, defensin and mytilin B), and 1 primer that will be used for the amplification of the house-hold gene. Selected enzyme Reference Mytilin A 82–84,180 Mytilin B 82–84,180 Mytilin C 82–84,180 Defensin 83 Peroxidase 81 Phenoloxidase 81 Lysozyme 68 Acidphosphatase 81 β-glucoronidase 81 Table III-2 The selected enzymes for gene expression analysis. B. Primer design All primers were designed, based on the coding DNA sequences (cds) of the gene of interest. It is important to use cds in this case because the primers are meant for conducting a reverse transcriptase real-time PCR in the future. These sequences were all requested from the NCBI- website (www.ncbi.com).181 If the exact sequence for the M. edulis gene of interest was found available, it was used for the design of the forward and reverse primers. The primer development was done with the Primer3-software182. Preferably, sequences were species specific for Mytilus edulis. If the specific coding DNA sequences from the gene of interest could not be found for the blue mussel, but it was available for at least two other species of the Mytilus genus an intermediate path was followed. In the latter case the BLAST tool was ran, to look for coding regions of high similarity between the two sequences of different, though related species. It was then this region which appeared to be quite conservative, that was used as a template for making the forward and reverse primers.

MATERIALS AND METHODS 35

The resulting primers were evaluated based on 3 characteristics; 1) Melting temperature of forward and reverse primer cannot differ more than 4°C, 2) the product size should be lower than 250 bp, and 3) forward and reverse primer cannot be similar in order to avoid formation of primer-dimer complexes.

C. DNA extraction Hemolymph samples were taken from the posterior adductor muscle of adult mussels with a 5ml syringe and a 23 gauge needle. 0.5ml of blood was taken per animal, and the blood of two animals was pooled. Between 0.08 and 0.12 gram of blood (~110µl) was weighted on a microscale (Sartonius, Van der Heyden). Each sample was mixed with 600µl of Nuclei Lysis solution (Promega-Wizard® Genomic DNA Purification Kit A1120), homogenised with a pestle for about 10 seconds, and subsequently incubated at 65°C for 15-30 minutes. Then 3 µl of RNase solution (Promega-Wizard® Genomic DNA Purification Kit, A1120) was added to remove RNA, and the sample was incubated for 15-30 minutes in the oven (Memmert, Analis) at 37°C. After incubation, 200 µl of protein precipitation solution was mixed with the suspension and the sample was then centrifuged for 4 minutes at 14 000 x g. The supernatant was transferred to a new eppendorf tube containing 600µl of isopropanol. This solution was centrifuged again for 1 minute at 15 000 x g and the resulting supernatant was discarded. The remaining DNA was washed with 70% ethanol and finally rehydrated in 50 µl of DNA rehydration solution (Promega- Wizard® Genomic DNA Purification Kit, A1120) overnight (at 4°C).

D. PCR immune genes Polymerase chain reactions (PCR) were performed with a MyCycler (Bio-Rad), in order to test the developed primers, and find the optimal annealing temperature for the immune gene amplification. Briefly, the PCR was executed in 25µl volumes containing; 2.5 µl of 10x Taq buffer (100mM Tris-HCl (pH 8.8)), 500mM KCl, and 0.8% (v/v) Nonidet P40) (Fermentas);

2.5µl of 25mM MgCl2 (Fermentas); 0.5 µl of 10mM dNTPmix (dATP, dGTP, dCTP and dTTP) (Fermentas); 0.25 µl of Taq polymerase (5U µl-1 (Fermentas)); 0.5µl of each primer at 10µM, 1 µl of undiluted DNA extract, and 17.25 µl of PCR water (SIGMA).

The amplification reaction consisted of 40 cycles, each cycle programmed for initial denaturation at 95°C for 4 min, followed by 40 cycles of denaturation at 94°C for 30 sec., primer annealing at variable temperatures for 1 min, extension at 72°C for 1 min., and a final delay step of 72°C for 5

MATERIALS AND METHODS 36

min. The different annealing temperatures that were tested according to this program are; 51°C, 55°C, 56°C and 60°C.

IV. Data analysis In a first place the assumptions for the parametric tests; 1) correlation of mean and variance, 2) normal distribution, 2) and homogeneity of the variances were evaluated. For the latter two assumptions the Shapiro test and Levene’s test was used respectively, while the firs assumption was evaluated visually. If the data was parametric, one- or multi-way anova analysis could be performed, followed by the Tukey HSD test to reveal the exact places of significant difference. In case of non-parametric data, the log-transformations or fourth root-transformations were applied and the assumptions were tested again. If the data did not fulfil to the assumptions, a Kruskal-Wallis test was performed, followed by a Wilcoxon test for post-hoc testing. All statistical analyses were executed with the R software package (version 2.14.1).

MATERIALS AND METHODS 37

PART IV. RESULTS

I. Development of a challenge test

A. Larval experiment The larval experiment has the aim

- to evaluate the material & methods on their usefulness for a challenge test - to screen 5 selected bacterial strains on their potential virulent effect on mussel larvae - to decide whether the addition of algae or trypton influences the larval survival/mortality rate

and was conducted in 2 phases:

First phase: control of bacterial strains

Prior to the screening test, the growth performance and possible contamination of the different strains was checked.

The origin of the bacterial strains that were used for this and following experiments varied from samples stored at the ARC stock over strains ordered from the LMG, and to self-isolated strains during the in vivo experiments. Simple growth experiments (on MA and TCBS) were performed initially to get an idea about the growth characteristics of the strains on respective media. The main finding was that strain LMG04437 was the only strain that did not grow on TCBS (annex 4, Table VIII-6). The results (annex 5, Figure VIII-1) of the pair-wise comparison of eight strains (api ZYM) did not indicate contamination. However, for all further tests it was decided to only use strains from the original stock and make them rifampicin resistant again. Nevertheless, it can be stated that the use of the api ZYM test (Biomérieux) is a reliable and fast technique to discern between different gram-negative bacteria.174,183

RESULTS 38

Second phase: screening test in quadruplicate Since no bacterial strain is described in literature suitable for a challenge test with M. edulis, 5 different strains with potential (annex 1,Table VIII-1) were selected for a first screening to evaluate their effect on larval survival. Three survival counts were made within a week after inoculation since this is the timing wherein a suitable challenge test should show its effect.

37 hours after challenge, the first set of samples was counted. The percentage mortality is summarised in Figure IV-1 below. First of all it has to be mentioned that unrealistic mortality values lower than 0% are the result of the error on the method of stocking at the start of the experiment (Annex 6, Table VIII-7 ) enhanced by the high survival rate.

A very high overall survival was seen in all the test wells. The three-way ANOVA did not result in a significant F-values when it comes to the interaction effect of the three independent variables; treatment, bacterial strain and bacterial concentration (F=1.173, p=0.317). Based on these results, none of the bacterial strains administered at a concentration of 105 or 106, did induced a significant mortality percentage, nor did the addition of LB or algae influence the results of the screening test , compared to the respective control group.

30 25

20 15 10

Mortality(%) 5 0

-5

Control Control Control

V. V. harveyiBB120 V. harveyiBB120 V. harveyiBB120

V. V. harveyi16835RIF V. harveyi16835RIF V. harveyi16835RIF

V. campbelli11257RIF V. V. V. campbelli11257RIF V. campbelli11257RIF

V. anguillarum4437RIF V. V. V. anguillarum4437RIF V. anguillarum4437RIF

V. alginolyticus4409RIF V. V. V. alginolyticus4409RIF V. alginolyticus4409RIF RIF RIF + Algae RIF + Trypton

Figure IV-1 Average mortality (%) of the larvae monitored 37 hours post IM injection.

RESULTS 39

Similar results were obtained after sampling at 61 hours post inoculation (Figure IV-2): no significant effect of the bacteria or treatment was seen (p=0.482, p=0.419). Also no significant interactive effect was seen between the bacteria on the one hand and the treatment on the other hand.

20

15 10 5

0 Mortality(%)

-5

V.… V.… V.…

Control Control Control

V. V. harveyiBB120 V. harveyiBB120 V. harveyiBB120

V. V. harveyi16835RIF V. harveyi16835RIF V. harveyi16835RIF

V. V. campbelli11257RIF V. campbelli11257RIF V. campbelli11257RIF

V. V. anguillarum4437RIF V. anguillarum4437RIF V. anguillarum4437RIF RIF RIF + Algae RIF + Trypton

Figure IV-2 Average mortality of the larvae recorded 61 hours p.i. After 83 hours there was no significant difference in mortality due to the effect of the challenged bacteria, within the three treatment groups (Figure IV-3).

25

20 15 10 5 0 -5 Mortality(%) -10

-15

Control Control Control

V. V. harveyiBB120 V. harveyiBB120 V. harveyiBB120

V. V. harveyi16835RIF V. harveyi16835RIF V. harveyi16835RIF

V. V. campbelli11257RIF V. campbelli11257RIF V. campbelli11257RIF

V. V. anguillarum4437RIF V. anguillarum4437RIF V. anguillarum4437RIF

V. V. alginolyticus4409RIF V. alginolyticus4409RIF V. alginolyticus4409RIF RIF RIF + Algae RIF + Trypton Figure IV-3 Average mortality recorded 83 hours p.i.

RESULTS 40

A small significant difference between treatment for the strain LMG16835 and the control group was detected (p= 0.039 and p=0.049). Nevertheless, it has to be kept in mind that these values are still quite high and the chance of making type I errors increases with the use of these non- parametric tests.

If the mortality between the three different sampling times is compared (37, 61 and 83 hours p.i.), we see significant differences among the groups that were only treated with rifampicin for all but one strain. This observation was not seen when the same analysis was performed on the other two treatments. When we have a closer look at all the samples which only received the rifampicin treatments, the calculated difference in mortality points have an illogic decreasing trend over time, which can be explained by the stocking error.

RESULTS 41

B. Adult experiments Instead of the in-bath treatment with the larvae, the pathogen was administered by means of intra- muscular injection in the posterior adductor muscle of the adult mussels.

The adult experiments have the aim

- To screen 7 selected bacterial strains on their potential virulent effect on adult mussels - To evaluate whether injected bacteria can be traced back in the hemolymph by plating - To evaluate whether the clearance rate in the hemolymph of the injected bacteria can be determined

- To evaluate whether the use MgCl2 described for oysters could also be used to sedate the blue mussel184,185

- To evaluate whether MgCl2 had influence on the mortality/survival rate for the time set of the pathogen screening test with IM injection

- To evaluate whether the exposure time of the blue mussel to MgCl2 has effect on the mortality/survival rate - To screen for potential effects of an enriched environment, physical stress of the host,

use of MgCl2, rifampicin water treatment that might help in the set up for a challenge test for M. edulis (annex 3 Table VIII-4 and Table VIII-5) and were conducted in three phases:

1) In vivo tests with bacterial strains 2) Anaesthesia experiment 3) Effect of enriched environment and physical stress on the host

RESULTS 42

1) In vivo tests with bacterial strains

Instead of the in-bath treatment with the larvae, the pathogen was administered by means of intra- muscular injection in the posterior adductor muscle of the adult mussels. The search for a suitable pathogen for a challenge test was continued. First a screening test was performed with 7 seven potential strains (the reason of their selection can be found in annex 1 Table VIII-1). The two most promising strains were then selected for a more complicated experiment in triplicate together with two fresh isolated strains from a mortality event in the stock.

a) Screening test with 7 Vibrio strains

Seven different Vibrio-strains were included in this screening experiment (annex 1, Table VIII-1), six known strains and one unknown isolate, previously isolated from tank water after a mortality event. The aim of this experiment was to verify whether 24 hours after IM injection, the bacteria strains could still be detected in the hemolymph by plating. Additionally, the mortality was monitored over a period of thirteen days. It should be mentioned that these animals did not get the rifampicin treatment before the onset of the experiment. 24 hours after injection the highest concentration of bacteria was found in the hemolymph of the mussels that were challenged with strain LMG11229RIF, followed by strain LMG16862RIF, LMG4409RIF and LMG16752RIF respectively (Table IV-1). The initial concentration administered IM to the mussels was 1x107 bacteria cells ml-1. The concentration detected by plating on MA with rifampicin (50 mg L-1) after 24 hours revealed that a maximum of 736 cells ml-1 could be found back in the hemolymph. Strain LMG11229RIF was therefore selected for the test in triplicate with multiple samples in time.

RESULTS 43

Bacteria code CFUs ml-1 Control 0 LMG04437RIF 0 LMG04409RIF 4 LMG16862RIF 252 LMG11229RIF 736 LMG16752RIF 4 BactXRIF 0 LMG10942RIF 0 Table IV-1 The average remaining bacterial concentration (CFU’s ml-1) per animal, challenged with the respective strain. The long-term mortality reached the highest value for those animals challenged with strain LMG10942RIF, LMG04437RIF and within the control group (Figure IV-4). All but one animal of the first group died, but more than 50% of this mortality started only ten days after the injection. Strain LMG4437RIF was therefore selected for the test in triplicate with multiple samples in time.

100,00 90,00 80,00

70,00 Control

60,00 04437RIF 50,00 04409RIF 40,00

%mortality 016862RIF 30,00 11229RIF 20,00 10,00 16752RIF 0,00 BactxRIF

10942RIF

0hp.i.

24hp.i. 72hp.i. 96hp.i.

48hp.i.

120hp.i. 144hp.i. 168hp.i. 216hp.i. 240hp.i. 264hp.i. 288hp.i. 312hp.i. 192h.p.i. hours post injection

Figure IV-4 Virulence of 7 Vibrio strains towards mussel adults.

RESULTS 44

Figure IV-5 describes the mortality outbreak that occurred within the batch of mussels still in stock, waiting to be used for the following experiment. The mortality was not recorded during the first days. The mussels to be used in the following experiment were treated with rifampicine from the third day onwards (when this high mortality was recorded), which successfully reduced number of dead animals during the following days, indicating a bacterial cause. From then on the mortality rate was recorded in both batches. Results for the batch without rifampicin is given in fig IV-5 Before an antibiotic treatment was started, water and tissue samples were taken. The on TCBS growing colonies were picked up, grown in LBmarine, and were made rifampicin resistant in order to use them in later experiments (In vivo test with 2ARIFResistant and 2Awildtype).This lead to the selection of the final two strains for the the test in triplicate with multiple samples in time.

50 45

40

35 30 25 20 Cum. Mortality (%)

15 Cum.mortality (%) 10 5 0

Figure IV-5 Pattern in mortality outbreak within the batch of stock mussels

RESULTS 45

b) In vivo test with LMG 11229RIF (V. tubiashii) and LMG4437 (V. anguillarum)

From previous screening test two bacteria strains were selected, strain LMG11229RIF due to its detectability in mussel hemolymph after 24hrs by plating (Table IV-1), and strain LMG4437RIF because it gave the highest mortality rate (Figure IV-4). The experiment was performed in triplicate and aimed to determine the clearance rate of the injected bacteria in the hemolymph over time. Additionally the mortality rate was monitored for 17 days. In contrast to the previous test, all test animals received a one-off rifampicin treatment (20 mg L-1 in tank water) before the experiment started.

The time lapse during which the challenged bacteria could be found back in the hemolymph by plating on MA (50 mg L-1 of rifampicin) is presented in (Figure IV-6). A slow, but steady decrease in bacteria within the blood of the animals that were challenged with LMG4437 can be observed. Strain 11229RIF was only found back within the blood at approximately 2 and 8 hours post injection. Less than 10 CFUs were counted on the control plates, within this time frame. The statistical analysis indicates that there was a significant higher concentration of strain LMG4437RIF in the blood of the challenged mussels compared to the controls at 0.5 and 2 hours post injection (p= 0.0369). Although a much higher bacterial concentration in the blood is also observed at one and four hours p.i., this could not be proven statistically, due to the fact that one of the replicates of the hemolymph samples did show very low growth after plating compared to the others.

10000,00

1000,00

Control 100,00 LMG 04437RIF

CFU/ml LMG11229RIF 10,00

1,00 0:39 1:02 2:10 7:55 Hours post injection

Figure IV-6. Trend in clearance of the bacteria out of the hemolymph.

RESULTS 46

During the first 48 hours the mortality increased up to 15% within the two groups that were challenged with the two bacterial strains, but the control group was also characterised with 10% of mortality (Figure IV-7). The mortality within the challenged groups reached a maximum of 20%, while within the control group it was 15% at the end of the experiment.

25%

20%

15%

10%

Mortality(%) Control 5% LMG4437RIF LMG11229RIF 0% 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 Hours post injection

Figure IV-7 Virulence of LMG4437RIF and LMG11229RIF towards mussel adults.

c) In vivo test with 2ARIFResistant and 2Awildtype

Since the mussel stock from the screening test suffered a high mortality (Figure IV-5), water and tissue samples were taken, and two morphologically different strains were isolated after growth on TCBS; 2A wildtype and 2B wildtype. The set-up of the previous clearance rate experiment and long-term in vivo experiment was repeated with rifampicin resistant strains of 2A and 2B (referred to as 2ARIF and 2BRIF). Despite our attempt to include both strains, 2BRIF did not grow well and was excluded from the test at the last minute. For the long term experiment 2ARIF, the animals received a one-off rifampicin treatment in advance (20 mg L-1) in the tank water.

An additional long-term experiment was set up, to test what would happen if no rifampicin would be used in an in vivo experiment. Here the test animals were not treated with the rifampicin in advance, and challenged with the wild type bacterium 2.A.

RESULTS 47

Unfortunately no useful results could be read from the plates and consequently no data can be reported about the clearance rate of the challenged bacteria 2ARIF. The MA-RIF plates inoculated with hemolymph taken at the start of the experiment, before any injection took place, indicated the presence of more than 3x103 CFU ml-1. Further, it was seen that the bacterial load in the undiluted samples of the challenged mussels was in the same order as in the corresponding control. This was true for each session of hemolymph collection. Since the hemolymph was plated on MA containing 50 mg/L of rifampicin, these observations suggest the presence of a rif- resistant bacterium, even before the onset of the experiment. Another later on proven possibility is that the rifampicin in the plates was not working anymore, allowing many bacteria to grow.

During the long term follow-up of the challenge with the rifampicin resistant strain 2.ARIF, no induced mortality was observed. It may thus be concluded that this strain is not virulent towards the blue mussel.

The animals that were challenged with 2A, and never received an antibiotic treatment suffered high mortality shortly after the challenge, within all replicates. However, it can be seen that the mortality within the control reached similar values (Figure IV-8). The experiment was performed over a period of eleven days, but all of the mortality took place within the first four days after injection.

120 100 80 60 Control 2.A 40 20

Cummulativemortality (%) 0 0 24 48 72 96 120 144 168 192 216 240 264 Hours post injection Figure IV-8 Virulence of wild type strain 2.A towards mussel adults.

RESULTS 48

2) Anaesthesia experiment

Since it is quite cumbersome to cut a hole in the shell of every single animal before injecting the mussel, another method was tested. Some authors refer to the use of MgCl2 as an anaesthetic for oysters, which relaxes the adductor muscles and consequently opens the valves. We successfully tried it on mussels and used it for al experiments where mussels were injected IM. To exclude an influence of the anaesthetic on the animals’ survival during these in vivo experiments, the effect of different exposure durations on the blue mussel was evaluated in a separate experiment by monitoring mortality for 48 hours, the duration of the experiments.

In all the experiments wherein MgCl2 was used to sedate the mussels ,a maximum exposure time of 2 hours was applied. No mortality was observed in this group during the first 48hrs of the anesthesia experiment. (Figure IV-9). Animals that were anesthetised for 8 consecutive hours showed a mortality rate of 5 % after 48 hours. Although the survival generally decreased 48 hours post challenge, no significant differences were detected between the different groups (p=0.1177). This means that the duration these exposures did not significantly affect the survival during the duration of the in- vivo-experiment.

25 Control

20 1 hr in anaesthetic

2 hrs in anaesthetic 15 3 hrs in anaesthetic

10 4 hrs in anaesthetic

Mortality(%) 5 hrs in anaesthetic 5 6 hrs in anaesthetic

0 7 hrs in anaesthetic 0 24 48 8 hrs in anaesthetic Hours post sedation Figure IV-9 Short-term effect of exposure time to the anaesthetic.

RESULTS 49

3) Effect of enriched environment and physical stress on the host None of the Vibrio strains in previous experiments induced significant mortality, and thus no pathogen favourable to be used in a challenge test for M. edulis was identified. During experiments performed earlier on, my supervisor observed high mortality in the experimental units that received significant higher doses of LB.

Therefore, the decision was made to start to play more with the factors environment (Rifampicin addition, enrichment with LB marine) and host (physical disturbance).

a) First screening

In this experiment the possible effect of stress, sedation and the admission of bacteria growth medium (LB) to the water was evaluated. The different treatments were combined in such a way that also the interaction between them could be followed (Annex 3, Table VIII-4). No extra bacteria were added in the water nor injected and the animals had the same origin of the mussels used in all previous experiments.

Figure IV-10 shows the effect of all the treatment combinations over an eleven-day period. The highest mortality was found with the animals that were shaked and got the LBmarine at the same time. The next groups with a final mortality ranging between 40-60% all received 1% LBmarine in their water. The groups with the best survival rate were the ones treated with rifampicin. This again indicates that the mortality rates are somehow related to the presence of bacteria.

When a high number of dead animals was found in a certain tanks, a water and tissue sample was taken. These samples were inoculated and grown overnight on TCBS plates and the colonies were picked up the next morning. These isolated bacteria were then further cultured in order to supplement the databank and later analyse their diversity.

RESULTS 50

100%

80%

60%

40%

Cum. Mortality (%) Mortality Cum. 20%

0% 1 2 3 4 5 6 7 8 9 10 11 Day

rifampicine + LB marine + shake rifampicine + LB marine rifampicine + anesthesia rifampicine + shake shake + anesthesia rifampicine rifampicine + shake + anesthesia LB marine + shake LB marine + shake + anesthesia anesthesia LB marine rifampicine + LB marine + anesthesia shake negative control LB marine + anesthesia rifampicine + LB marine + shake + anesthesia

Figure IV-10 Mortality during the firs 11 day screening experiment.

b) Second screening

Based on the previous screening experiment it seems that LB addition and shaking were the factors that resulted in the highest mortality. These factors where thus selected for the next experiment in triplicate., again without adding any extra bacteria strains besides the ones naturally present from the start of the experiment. A negative (no rifampicin treatment) and a positive control (rifampicin treatment) group was included. (Table VIII-5).

Figure IV-11 clearly shows an increasing mortality within the groups that were treated with LBmarine only, and those that were shaked and received LBmarine at the same time. LB treatment resulted in a significant higher mortality at the end of the experiment compared to the negative and positive control (reps. p= 0.006 and p=0.005), and the variation among replicates was also quite low. The same is true for the animals treated with LBmarine+ shake (p=0.006 and p=0.005). However no significant difference was seen between LBmarine and the LBmarine+shake treatment (p=1), neither between the shake-treatment and the negative and

RESULTS 51 positive controls (p=0.80 and 0.73). This indicates that the high mortality in group LBmarine and LBmarine +shake, can be fully attributed to the effect of the addition of LBmarine.

100 90

80

70 60 50 40

30 cum.mortality (%) 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 Time (days) negative control positive control (with rifampicin) LB + shake LB shake Figure IV-11 Mortality during second screening test.

RESULTS 52

C. Diversity of the isolated strains

During the in vivo experiments of this study, 57 strains were isolated from the tank water, and dead or moribund animals (Annex 3). To get a first idea of the diversity within this group of samples, an ERIC-PCR was performed to give a specific DNA fingerprint for each isolated strain. Several PCR’s were run in which strain BB120 was incorporated, since the ERIC-PCR finger print is known for this strain.178 The ones with annealing temperature of 45°C and 51 °C gave the following result (Figure IV-12).

Figure IV-12 icture of agarose gel (A.G.) electrophoresis. L: Ladder, NC: negative control. Left A.G.: PCR with annealing temperature of 45°C. E1: cell extract (100x); E2: DNA extract (0x); E3: DNA extract (100x). Gel was run at 75V for 70 min. Right A.G.: PCR with annealing temperature of 51°C. DNA extract 0, 100 and 1000 times diluted. Gel was run at 100V for 35 min. The left gel shows that the lowest primer concentrations (0.5µM) results in the best amplification of the target sequences (Figure IV-12). From the two different DNA extractions that were used (boiling washed cell vs. Promega kit), the DNA extracts obtained via the Promega DNA extraction kit (E2 and E3) gave a slightly better image. Despite the successful amplification of

RESULTS 53 target sequences, an excessive amount of smear in all lanes darkens the view and makes the interpretation quite hard.

The PCR that was run at an annealing temperature of 51°C did not work optimally, although a decreasing trend in smear is seen when a lower DNA concentration is used (Figure IV-12). Also one trial was performed with a shorter program and an annealing temperature at 56°C, but this did not result in any amplification at all.

RESULTS 54

II. Development of a molecular toolbox to study the expression of immune genes

The second part of this thesis aimed at developing a toolbox to investigate the reactions of the immune system of the blue mussel after challenge with a pathogen, by reverse transcriptase realtime PCR with specific primers for different immune genes. Selected genes included defensin, mytilin and lysozyme. The 18S rRNA gene was selected as housekeeping gene and primers were taken from literature.91 Hemolymph samples were collected during in vivo experiments with adult mussel.

A. Primer Design

The sequences that were selected for the primer design of lysozyme, mytilin B and defensin can be seen in Figure IV-13. Regarding mytilin B the template was a highly conservative sequence (partial cds) of M. galloprovincialis, identified after a sequence alignment with M. coruscus, mytilin B (partial cds). Regarding defensin, the highly conservative region was identified after sequence alignment of M. chilensis (partial cds) with M. galloprovincialis (partial cds), and the former one served as the template. The lysozyme primers were designed based on M. edulis coding DNA, which was available in the genbank (www.ncbi.com).

Figure IV-13 Sequences for primer design. >>>>>>: Left primer (forward), <<<<<<: right primer (reverse).

The primers can be found in Table IV-2. Additionally, the primers for the housekeeping gene are listed in this table as well. The latter one was found in an article of Espinosa et al (2010)80,

RESULTS 55 where it had been found suitable for reverse transcriptase real time PCR. This table also summarises main characteristics of the primers as they were given by the company that made the primers (Eurogentec).

Oligo Sequence Bases Tm %GC amplicon length (bp) in reverse name transcriptase RT-PCR DefMytF GAA-AGA-AGA-AAA-GCG-GTA-TGC 21 45,3 42,9 104 DefMytR AAG-CGC-CAT-ATG-CTG-CTA-CT 20 60 50 LysMytF GTT-TAC-TGG-GAT-GGC-TGT-GG 20 62 55 212 LysMytR TGA-ATA-TGG-CTC-CCG-TAA-CC 20 60 50 MytilinF TGT-AGA-GCA-AGA-CGC-TGT-GG 20 62 55 210 MytilinR TCA-CCT-TGT-TCG-GTT-TCT-CC 20 60 50 18SMytF CTG-GTT-AAT-TCC-GAT-AAC-GAA- 31 55,3 41,9 181 CGA-GAC-TCT-A 18SMytR TGC-TCA-ATC-TCG-TGT-GGC-TAA- 31 59,3 51,6 ACG-CCA-CTT-G Table IV-2 Sequence characteristics of the primers according to EuroGentec. GC: Guanine-Cytosine. bp: base pairs. Tm: melting temperature.

B. DNA extraction

The hemolymph was collected from mussels of the in vivo experiment and used for DNA extraction. The 18S rRNA gene could be amplified from all three extracts tested, giving a fragment with the expected length, however sample 1B (1) showed some degree of DNA degradation (smear pattern) (Figure IV-14). This extract was not used for further PCR reactions.

RESULTS 56

Figure IV-14 Picture of agarose gel with DNA extract of mussel hemocytes (left) and PCR amplicons of the 18S rRNA gene. NC: negative control, L: DNA ladder (80-1031 bp).

C. PCR immune genes

Subsequently, the immune genes were tested in 4 PCR reactions, using different annealing temperatures (i.e. 51°C, 55°C, 56°C and 60°C respectively) (Figure IV-15, A-E). The 18S rRNA gene was amplified in all four PCRs. The specific primers for the defensin gene did also succeed in amplifying a product, with a length of 104 bp (Table IV-2) at all four annealing temperatures tested. The specific primers for the mytilin B gene, however, did show two bands on the gel, one of +400 bp and another one of +700 bp in length (Figure IV-15A,D,E). In contrast, the specific primers for the lysozyme gene did not produce an amplicon in any of the PCRs that were run during this thesis (Figure IV-15A,D,E). Explanations for the observation regarding mytilin B and lysozyme will be given in the discussion of these results.

RESULTS 57

Figure IV-15 Picture of agarose gels with PCR-products of the immune gene primers. NC: negative control, L: ladder.

RESULTS 58

PART V. DISCUSSION

I. Development of a challenge test

The aim of the in vivo experiments was to develop a standardised protocol for a challenge test for M. edulis larvae and to set the first steps towards further research on host-pathogen interaction.

Regarding the development of the challenge test, two phases can be distinguished:

- set up of a methodology protocol - selection of a suitable pathogen (one that causes repeatable stable mortality among the test animals).

In following chapter an evaluation of the methods that were used for the larval and adult tests was made, together with the identification of the critical points which were encountered. Finally a detailed analysis was made on the different strains that were used.

A. Evaluation of the methodology

1) Larval experiment

Working with larvae varying in length from 30-90 µm has its limitations regarding handling procedures and accurate mortality evaluation. If we look back at the methodology of this larval challenge experiment, several comments can be given on the way this test was executed and the materials that were used. The 24-well plates, used to incubate the larvae were found suitable for the challenge test, as other authors confirm.107,119 Their limited size allowed to monitor the mortality accurately and to work in a confortable way in the laminar flow. No larvae got lost by sampling actions since complete counts were made. Sandlund et al. (2006) restricted the larval density to 20-40 larvae ml-1. Looking at the very high survival rate in our experiments with stocking densities of 250 larvae ml-1 (over ten times higher) one can conclude that this restriction should be reconsidered.

Counting the larvae for assessing the survival was done after lugol staining. In literature, researchers however count the larvae when they are still alive.106,107,119 The advantage of lugol staining is that the counting can be performed more precisely because the larvae are immobile

DISCUSSION 59 and well visible under a binocular (stained dark brown). One cannot exclude that recently dead larvae are stained as well, but since degradation happens very fast in the aquactic environment and counts were made on consecutive time sets this should be of no influence for our setup. A motif for life-counting mentioned in literature is that the discrimination between live, moribund/abnormal and dead larvae can be made, and that certain disease symptoms can be studied more precisely. 106 However since M. edulis larvae at this early life-stage do not swim actively all day long and have natural periods of rest on the bottom one could ask himself whether they can be considered as moribund. One big disadvantage of lugol staining in contrast to live counts is that the larvae needed to be sacrificed at every counting moment. On the other hand, lugol staining allowed evaluating mortality of all test wells within a limited time. The larvae preserve well after lugol addition, whereas life-counting requires more time, and thus a time interval between parallel samples seems unavoidable.

The addition of trypton or algae did not affect the survival of the larvae. It was expected that the trypton addition would stimulate bacterial growth in the wells, since it is a source of amino acids.186 Whether bacterial growth was stimulated or not cannot be confirmed, but no increased mortality was detected in any of the wells. In addition in should be kept in mind that bacteria can also be a food source to the larvae, and therefore have a positive effect on larval survival. 89,187 Whether the algae or bacteria beneficially affected the survival of the 2 to 5-day old larvae cannot be confirmed either with the results of this experiment, since survival was nearly 100% in all treatments. Although veliger larvae are capable to eat from the prodissoconch I stage (D- stage)188, this experiment confirmed that mussel larvae can survive quite long periods of starvation.

2) Adult tests

The unavoidable shift from larvae to adults due to the season was evaluated positive since it gave the opportunity to administer the potential pathogens intramuscularly.

The challenge by means of IM injection shows to be quite reliable in administering a known dose of bacteria. It requires some practise to become familiar with finding and penetrating into the

DISCUSSION 60 posterior adductor muscle, but once the necessary experience is acquired it is a fast and effective method. A draw-back of the injection in the muscle is that it might weaken the animal, and wounding the muscle may increase the risk of secondary infections. Another disadvantage is the uncertainty of the physiological effect of the anaesthetic, which will be discussed later on. Nevertheless, it is a technique which has shown its usefulness in previous experiments.93,130 Hemolymph collection from the posterior adductor muscle is also quite convenient. Some authors withdraw hemolymph from the pericardial cavity, but this seems more complicated.93

The enumeration of the bacterial load in the hemolymph by plating the hemolymph sample was successful. The low percentage CFUs on the MA-RIF compared to the administered dosage could be explained by dilution and one should keep in mind that not every bacteria present will effectively grow on an agar plate. The results of the challenge experiment with 2.ARif indicated the presence of many different bacteria. This was probably caused by break-down of the rifampicin inside the agar due to the storage of the plates in an illuminated room,.

Not many problems were associated with the long-term follow up of the adult animals after challenge. In the beginning of an experiment the amount of faeces in the boxes was higher than after some days of starvation. In the future a longer period of quarantine can be considered as a safety measure, because mussels are capable to bio-accumulate bacteria, and concentrate high loads of bacteria in their faeces.189 Nevertheless, we did not encounter many problems with this, and neither ammonia, nor nitrite levels ever reached worrying levels (Annex 7: Table VIII-8 and Table VIII-9) because the water was changed on a daily basis. The disadvantage of this daily water refreshment is that it increases the risk on contamination.

The chemical MgCl2 effectively relaxes the adductor muscles of bivalves and by doing so it has already been a great help for research and industry in other bivalve species but was never studied on M. edulis before. 117,176,184 Based on the results from the anaesthetic experiment we can conclude that MgCl2 can be a valuable tool for M. edulis, in order to facilitate the injection and hemolymph collection process. The treatment with this chemical does not induce a significant mortality compared to the control, and neither does the duration of the anaesthetic exposure has any negative implications. This determination coincides with what was previously observed in other bivalve species e.g. C. gigas, O. edulis and P. fumatus.175,184,185

DISCUSSION 61

-1 The dose of 28 g L (0.3 M) of MgCl2 which is applied in my experiment is slightly lower than which can be found back in literature. To illustrate, some authors used concentrations of 30 g L-1, 32 g L-1 up to 72 g L-1. 175,176,184,185,190 Nevertheless, the speed-efficacy which I observed, namely 100% sedation after 1 hour, is a quite good score when it is compared to those studies. Temperature can play role in the rate at which anaesthesia develops, but this effect might be species dependent since contradictory statements were found in literature. 175,184,185

A critical note should be made when MgCl2 will be used in future challenge experiments. It is known that Mg2+-ions inhibit neuronal communication at the level of the synapses, and in such a way they are responsible for the relaxation of the adductor muscles of bivalves. Besides this knowledge on the mode of action of this chemical, there is not so much known on other physiological effects. Butt et al. (2007)191 reported that several stress indicators of the Sydney rock oyster (S. glomerata), e.g. total hemocyte frequencies, acid phosphatase, superoxide and phenoloxidase activities, significantly increased due to exposure to MgCl2. Careful attention has to be paid if this anaesthetic is used in a challenge test where similar stress indicators want to be used to monitor the effect of a bacterium. It is advisable to perform preliminary experiments to assess the effect of MgCl2 on Mytilus edulis, in order to study the physiological effects of this chemical.

The main result of last two in vivo experiments performed to evaluate the possible influence of the environment (Effect of enriched environment and physical stress on the host) is that groups receiving the LB marine treatment were characterised by a higher incidence in mortality. On first sight two plausible hypotheses can explain this observation; or the broth itself is toxic or this medium favoured the growth of pathogens, and in such a way it is indirectly responsible for the high mortality percentages. The former explanation may seem quite unrealistic since the basic composition of LB broth would not suggest any harmful effect of this product.192 However, Luna-Gonzalez et al. (2002)105 stated that trypticase soy broth (TSB) showed signs of toxicity towards bivalve larvae during their preliminary experiments. Generally, TSB and LB broth do only differ in their source of amino acids, which is soytone vs. yeast extract respectively. Although we cannot exclude that LB broth by itself will affect mussel larvae, it seems unlikely that it is responsible for the dead of the adult mussels. The second hypothesis, which attributes the mortality to the indirect effect of (an) opportunistic pathogen(s), is therefore the most realistic

DISCUSSION 62 one. Moreover, this statement can by reinforced by the observation of high survival of those animals that were treated with LB marine in combination with the antibiotic rifampicin.

The last handling that was evaluated is the shaking method, or in other words the effect of a physical disturbance of the mussels (most likely inducing stress) on their survival. The second screening test clearly shows that shaking on its own did not result in significant higher mortality percentage of the animals, compared to the controls. The slightly higher mortality within this group can be explained by the fact that some animals’ shell was damaged after the two minutes of shaking, resulting in a possible port of entry for pathogens. Nevertheless, the mortality probably remained relatively low thanks to the high natural resistance of the blue mussel against physical disturbances. An experiment by Bussel et al (2008) also could not prove any deleterious effect on the metabolism of mussels, caused physically disturbing the animals, and thus partly supports previous statement. 167

B. Discussion of virulence of the tested strains Of the eleven strains which were tested during the experiments, no bacterium showed significant virulence towards the blue mussel, Mytilus edulis. Nine strains belonged to seven different Vibrio species: V. harveyi, V. alginolyticus, V. campbelli, V. splendidus, V. proteolyticus, V. tubiashii and V. anguillarum, while the two remaining strains (2.A and Bact.X) were not identified yet since they were isolated from dead animals during the experiment. The origin of the nine identified strains is quite diverse; some have been isolated from sea water, others from diseased fish, prawns or bivalves (Table VIII-1). However, no reference was found that indicates individual virulence towards the blue mussel. Only for the strains LMG 4409, LMG10942 and LMG4437 it was reported that they induced a certain reaction in M. galloprovincialis, O.edulis and M. galloprovincialis respectively.106,145,148 Since it is known that the virulence of a Vibrio strain can be very host specific, the lack of induced high mortality during our experiments is not so abnormal. On the other hand, the results of the challenge with BactX and mainly strain 2.ARIF did not follow our first expectations. These strains were isolated after sudden mortality events that occurred during the in vivo experiments of this thesis. However it is also possible that the isolated bacteria were just the most abundant ones, but not necessarily the pathogens. It is also possible that more than one strain (or one factor) was responsible for the mortality event. The fact that the

DISCUSSION 63 experiment with the wild type of strain 2.A did result in high mortality in both the challenged groups and the controls suggest that a pathogen was present in the water. One possibility is that the strain 2.A is responsible for the observed mortality in the control group, due to contamination from the tanks with the challenged animals. The tanks were located in each other’s vicinity, which may have caused cross-contamination. The other possibility is that the pathogen came along with the animals, and since they were not treated with the antibiotic in advance, it had the chance to develop due to favourable growth conditions (e.g. low water renewal rate, high nutrient concentrations;…). Forthcoming from the result of this last experiment, some additional literature research was done to find out what is known about side effects of rifampicin on resistant bacteria. As I mentioned before, using a resistant strain instead of the wild type, gives us the advantage that an antibiotic can be used during the challenge, and the risk of contamination by an unknown pathogen can be minimised. Nevertheless, it is interesting to think about possible consequences such an acquired resistance might have on the bacteria’s behaviour. It is known that antibiotic resistant mutants can have a lower competitive fitness compared to their wild type strain.193,194 Several researches have proven that this is also true for mutants that became resistant to rifampicin, and a consequential loss of virulence in the mutant strain has been determined as well.195–201 The degree of the additional fitness burden however varies with the exact location of the RNA polymerase (rpoB) mutation.195,200 It was also seen that after subsequent isolation-injection steps in vivo, or several following culture steps in vitro, compensatory mutations can take place which ameliorate the initial fitness loss.196–198 These papers all refer to bacteria species other than Vibrio spp. e.g. Salmonella spp., E. coli, S. aureus, M. tuberculosis; … This does not imply that the side effect of rifampicin resistance would be similar for the Vibrios used in this thesis, however the point mutation that will give resistance to a mutant occurs within the same gene. Careful attention has to be paid when rifampicin is used in challenge tests. In conclusion, it should be kept in mind that absence of mortality can be a consequence of; 1) the good working immune system of the mussel, 2) the lower fitness of the rifampicin resistant mutant, 3) or the combination of these two things.

DISCUSSION 64

C. Diversity of the isolated strains

During this thesis I did not get to the actual analysis of the isolated strains using the ERIC-PCR. Nevertheless, the several runs that were executed did reveal useful information. First of all it was shown that the lower primer concentration of 0.5 µM results in a slightly better amplification of the targets than the high concentration of 2µM that was prescribed by Ruwandeepika et al. (2010) (Figure IV-12).178 Secondly, the lower DNA-concentrations did result in significant lower amounts of smear on the gel. Several hypotheses could explain this smear. In theory, it can be a consequence of degraded DNA, due to unfavourable storage conditions. This however is quite unlikely since the original DNA sample did not show the smear when it was loaded on the gel, and the PCR products were immediately frozen after completing the program. A second explanation might be that the high concentration of DNA, in combination with the low annealing temperature. Regarding the annealing temperature it is generally known that the specificity of a PCR lowers significantly when the more the annealing temperature decreases below the optimal annealing temperature of the primers.202 This hypothesis is supported by the observation that the PCR ran at an annealing temperature of 45°C resulted in more smear than the one ran at the near- optimal temperature of 51°C. Moreover, the latter PCR showed that lower DNA concentrations resulted in lower amounts of smear. Finally it should also be mentioned that these two ERIC- PCRs were also used to test the immune genes, and their products as well showed an excessive amount of smear when loaded on the gel, also in their negative controls. When the immune genes were tested in a shorter program with annealing temperature of 51°C this smear was not seen. This suggests that the duration of the elongation step might be too long (10 min), although the program is similar as the one other researchers used. 178,179

DISCUSSION 65

II. Development of a molecular toolbox to study the expression of immune genes

The results in Figure IV-14 indicate that the DNA extraction protocol which was followed ( DNA extraction) is suitable for the DNA extraction from M. edulis hemocytes. Since some samples did contain a lower amount of DNA, the consideration can be made to increase the amount of sampled tissue (blood cells). In this case, about 110 µl was used, but this volume can be increased up to 500 or 1000 µl. To overcome the problem of degenerated DNA, the final drying step of the extraction process could be shortened or other storage conditions (e.g. Tris-EDTA buffer or trehalose) could be considered.203

The primers of the 18S ribosomal RNA gene were tested and did successfully amplify the target sequence of 181 bp. These primers were taken from Espinosa et al. (2010), who used an annealing temperature of 50°C.91 The test results of this thesis show that the annealing temperature can be increased up to at least 60°C,.202 18S ribosomal RNA forms part of the small subunit of eukaryotic ribosomes, which as a whole are responsible for the translation of messenger RNA. Since these macromolecules are always present in the cell, the transcription of the sequence of the genes coding for every sub-unit is assumed to take place at a steady rate.204 Because the expression rate of this sequence is quite stable, we can use it as a control reference for monitoring the expression level of immune related genes which might change due to the presence of a bacteria.66,83 Given the positive results, this primer pair can be taken up in a real- time PCR trial.

The primers for the defensin gene were not designed based on an existing M. edulis sequence; a region of a high similarity between the defensin sequences of M. chilensis on the one hand, and M. galloprovincialis on the other hand, (identified with the BLAST program at www.ncbi.com) were used. The successful amplification of the target, suggests that this sequence is highly conserved among at least three Mytilus species; M. edulis, M. galloprovincialis and M. chilensis. Since the relatedness of European Mytilus spp. has been mapped already, and the Chilean mussel is considered as a subspecies of Mytilus edulis by some but not all authors, this finding is realistic.205–207

DISCUSSION 66

The primers designed for the mytilin B gene did amplify two fragments; one of +400 bp and one of + 700 bp. However, a decreasing amount of the smallest amplicon is seen when a higher annealing temperature is applied. This suggests that the smallest fragment is the result of nonspecific binding of the primers, and that this signal might disappear when a higher annealing temperature is used. When we have a look at the genome of the closest neighbour of the blue mussel, M. galloprovincialis a possible answer can be formulated. Annex 9 (Figure VIII-3) shows the result of a sequence alignment that was run with the ClustalW2 tool of the European Bioinformatics Institute (www.ebi.ac.uk). This multiple alignment included the genomic sequences of the Mytilin B gene on the one hand, and the sequence that was used for the primer design on the other hand. If the regions where the primers should anneal are traced (when the genomic DNA of M. galloprovincialis is used), an intron of 517 bp can be seen in between the forward and reverse primer. In total, this would result in an amplification of a fragment of 777 bp. This observation strongly suggests that the amplicon of + 700 bp that was seen on the gel, is the real product and the smaller one is a nonspecific band.

Although the lysozyme primers were the only ones developed based on the Mytilus edulis sequence, no amplification was observed. The explanation for this can be seen in annex 9 (Figure VIII-2). This similarity profile was obtained by running the Multiple Sequence Alignment tool (ClustalW2, www.ebi.ac.uk), including the complete genomic DNA of lysozyme on one side and its partial cds, that was used for the primer design, on the other hand. It can be seen that in between the two, genomic binding sites of the primers about 889 bp of non-coding DNA (introns) is located. Due to the fact that genomic DNA was used for these test, the presence of the intron makes that the amplicon has a total length of 1128 bp. However, the PCR protocol that was applied did not anticipate on such a large fragment, and the extension step of 1 min. might have been too short. Extending the elongation step is one option, another option is using to this primer pair.

In summary, it can be concluded that both the 18S rRNA and the defensin primers are suitable for the real-time PCR. Further testing with higher annealing temperatures for the lysozyme and mytilin B primers will reveal if previously made hypotheses are correct, and if the respective primers can be used as well or not. Finally, the results of the mytilin B and defensin tests suggest that the host-pathogen interaction that lies at the basis of the selective evolution towards these

DISCUSSION 67 two antimicrobial peptides, took place before the geographic isolation of at least four Mytilus sp.(i.e. M. edulis, M. galloprovincialis, M. coruscus and M. chilensis).

DISCUSSION 68

PART VI. CONCLUSION AND RECOMMENDATIONS FOR THE FUTURE

First of all the new strains that are isolated, have to be processed in an ERIC PCR in order to reveal the diversity among them. Since the DNA extraction is already performed during this thesis (Annex 8,Table VIII-10), only further dilutions (e.g.1/1000) have to be made. When the independent strains are then identified, the bacteria can be taken up in the next in vivo experiments, and the quest for a M. edulis pathogen can be continued. It should be considered to perform in vivo experiments with several bacteria together, since interaction between them may be crucial to develop virulence. Regarding the protocol of the in vivo experiments, following optimisations are recommended. The way the larval test was performed, is quite acceptable when compared to other methods described in literature. The only step that needs to be optimised is the mixing process before dividing the larvae among the test plates. Maybe preliminary tests with magnetic stirrers can be done to see if this results in a better homogenisation of the larvae in suspension in order to uniform the stocking ratio. The adult test performed during this thesis, were done for the first time so it is clear that the set- up can still be improved. Special attention should be paid to the reduction of risk of contamination. A separate recirculation system for each experimental unit can make things easier and will also lower the chance of making mistakes during daily handling procedures. In general, the last two experiments revealed that shaking did not induce significant higher mortality to the animals, so this method can still be applied, although the actual sense of this step can be discussed. The same experiments did show that the presence of 1% LBmarine probably results in a better growth of bacteria, with a higher mortality as a consequence. The addition of LB marine in tests that aim to find a suitable pathogen for a challenge test, is therefore advisable. Finally, the use of MgCl2 was evaluated positively as well, although the potential side effects might have to be investigated more in detail, as mentioned in the discussion. The analysis of the host-pathogen interaction, with the bacterial clearance rate and the additional long-term survival as the main interaction parameters, is now standardised quite well. In future experiments these two methods of evaluation can be applied, in combination with a more detailed study on the immune system, via a real time PCR. The primers for this gene expression analysis have been developed during this work, and two pairs have been proven to amplify their target CONCLUSION AND RECOMMENDATIONS FOR THE FUTURE 69 successfully. Since the primers of the house-hold gene 18SrRNA on one hand, and those for the immune gene defensin on the other hand both work up to an annealing temperature of 60°C, they can be used in one PCR-run. Finally, the search for the optimal binding temperature of the other primers should be continued. As discussed before it is quite likely that the primers of the defensin gene will generate a single amplicon if the annealing temperature of the PCR is increased with a few degrees. Regarding the lysozyme primers, a PCR program with a longer elongation step and an annealing temperature of 60-65°C will confirm or refute previously made hypothesis. If the hypothesis is incorrect, new primers can be designed for this lysozyme gene. Additional, immune genes can be selected as well for real time PCR assays. The results of defensin and mytilin B indicate high conservativeness of these genes, so if primers have to be made in the future it can be considered to use genomic DNA of M. galloprovincialis from the start. The more immune genes are included in the gene expression analysis, the better the understanding of the immune system of the blue mussel, Mytilus edulis.

CONCLUSION AND RECOMMENDATIONS FOR THE FUTURE 70

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PART VIII. APPENDIX ANNEX1 Information on bacterial strains used during this thesis

APPENDIX 86

Challenge Lab name Species Isolated from experiment Ref. Observation BB120 V.harveyi / larvae 147 Effect on mortality and V.alginolyticus spoiled horse mackerel normality of M. 146

LMG 4409 (Trachurus trachurus) larvae, adult 1 galloprovincialis larvae; V. harveyi LMG 16835 Black Tiger Prawn, Thailand larvae

LMG 11257 V. campbelli Sea water, Hawai larvae V. campbelli LMG 11216 Sea water, Hawai V. splendidus LMG 16752 Oyster, Spain adult 1 LMG16862 V.hareyi Oyster spain adult 1 hard clam (Mercenaria V.proteolyticu mercenaria), moribund larvae, s 148

LMG 10942 USA adult 1 Proteinase toxic to O. edulis moribund juvenile oyster larvae, adult V. tubiashii

LMG 11229 (Crassostrea virginica) 1&2 cod (Gadus morhua), ulcerous Effect on haemocytes of adult L.anguillarum 67

LMG 4437 lesion, Norway adult 1&2 M. galloprovincialis Culture water of dead mussel BactX N.I. larvae, ARC adult 1 Culture water moribund/dead 1A N.I. adult mussels, ARC Culture water moribund/dead 1B N.I. adult mussels, ARC Culture water moribund/dead 2A N.I. adult mussels, ARC adult 3 Culture water moribund/dead 2B N.I. adult mussels, ARC Hemolymphe moribund/dead JanIa(1) N.I. adult mussels, ARC Hemolymphe moribund/dead JanIb(1) N.I. adult mussels, ARC Hemolymphe moribund/dead JanHa(1) N.I. adult mussels, ARC Hemolymphe moribund/dead JanHb(1) N.I. adult mussels, ARC Hemolymphe moribund/dead JanHa(2) N.I. adult mussels, ARC Hemolymphe moribund/dead JanHb(2) N.I. adult mussels, ARC Table VIII-1 Summary of the bacterial strains used in each experiment, with the corresponding origin of the strain. Larvae: strains used in first larval experiment. Adult 1: strain used in “General screening of 7 Vibrio strains, Adult 2: strain used in “In vivo test with LMG 11229RIF (V. tubiashii) and LMG4437 (V. anguillarum). Adult 3: strain used in “in vivo testwith 2ARIFresistant and 2Awildtype”. N.I: Not identified. ANNEX 2 Injection and hemolymph collection scheme

APPENDIX 87

Table VIII-2 Injection (INJ) and hemolyph collection (COLL) moments for the in vivo test with LMG4437RIF and LMG 11229RIF.

Table VIII-3 Injection (INJ) and hemolymph collection (COLL) moments for the in vivo test with 2ARIF.

APPENDIX 88

ANNEX 3 Treatment characteristics during the test “Effect of enriched environment and physical stress on the host”

Treatment Tank Code Rifampicin LB marine Shake Anaesthesia A + + + - B + + - - C + - - + D + - + - E - - + + F + - - - G + - + + H - + + - I - + + + J - - - + K - + - - L + + - + M - - + - O - + - + P + + + + Table VIII-4 Treatment combination characteristics per tank during the first screening test.

Treatment Tank Code rifampicine LB marine shake 1a - - - 1b - - - 1c - - - 2a + + + 2b + + + 2c + + + 3a - + + 3b - + + 3c - + + 4a - + - 4b - + - 4c - + - 5a - - + 5b - - + 5c - - + Table VIII-5 Treatment combination characteristics per tank during the second screening test.

.

APPENDIX 89

ANNEX 4 Growth performances of bacteria used during this thesis

StrainCode Species MA TCBS Colony morphology BB120 Vibrio harveyi + + Ma: White – TCBS: Pale green BB120RIF Vibrio harveyi + + Ma: White – TCBS: Pale green LMG04409 Vibrio alginolyticus + + MA: white - TCBS: yellow LMG04409RIF Vibrio alginolyticus + + MA: white - TCBS: green LMG16835 Vibrio harveyi + + MA: white – TCBS: green center, light periphery LMG16835RIF Vibrio harveyi + + MA: white – TCBS: green center, light periphery LMG11257 Vibrio campbelli + + MA: white – TCBS: green LMG11257RIF Vibrio campbelli + - MA: white – TCBS: / LMG11216 Vibrio campbelli + + MA: white – TCBS: green LMG11216RIF Vibrio campbelli + - MA: white – TCBS: LMG16752 Vibrio splendidus + + MA: white – TCBS: dark green center LMG16752RIF Vibrio splendidus + + MA: white – TCBS: dark green center LMG10942 Vibrio proteolyticus + + MA: white – TCBS: pale green LMG10942RIF Vibrio proteolyticus + + MA: white – TCBS: pale green LMG11229 Vibrio tubiashii + + MA: white – TCBS: yellow LMG11229RIF Vibrio tubiashii + + MA: white – TCBS: yellow LMG4437 Listonella anguillarum + - MA: white – TCBS: LMG4437RIF Listonella anguillarum + - MA: white – TCBS: BactX N.I. + + TCBS: Yellow BactXRIF N.I. + + 1A N.I. + + 1ARIF N.I. + + 1B N.I. + + 1BRIF N.I. + + 2A N.I. + + 2ARIF N.I. + + 2B N.I. + + 2BRIF N.I. + + JanIa(1) N.I. + + JanIa(1)RIF N.I. / / JanIb(1) N.I. + + JanIb(1)RIF N.I. / / JanHa(1) N.I. + + JanHa(1)RIF N.I. / / JanHb(1) N.I. + + JanHb(1)RIF N.I. / / JanHa(2) N.I. + + JanHa(2)RIF N.I. / / JanHb(2) N.I. + + JanHb(2)RIF N.I. / / Table VIII-6 Evaluation of the growth after 24 hours of incubation (30°C). N.I.: Not identified; /: Was not made resistant because it had never been used in an experiment yet.

APPENDIX 90

ANNEX 5 Results of api ZYM tests

Figure VIII-1Result api ZYM test to compare wild type strains with their respective mutant strains.

APPENDIX 91

ANNEX 6 Average density of larvae per 24-well plate.

Plate Average larval Time of counting (hrs Exposure density (cells ml number density (larvae ml-1) p.i.) -1) 1 266 (+17) 37 105 2 247 (+14) 61 105 3 244 (+10) 83 105 4 251 (+11) 37 106 5 242.5 (+1) 37 105 6 237.5 (+1) 61 105 7 228 (+16) 83 105 8 232 (+9) 37 106 9 270 (+10) 37 105 10 238 (+14) 61 105 11 266.5 (+12) 83 105 12 243 (+8) 37 106 Table VIII-7. Information on each plate used during this experiment. 1-4: Only rifampicin, 5-8: Rifampicin + algae, 9-12: Rifampicin + Trypton.

APPENDIX 92

ANNEX 7 Water quality during the tests to evaluate the effect of enriched environment and physical stress of the hosts.

Sampling Tan Nitrite (mg Ammonium (mg Oxygen (mg Temperature time k L-1) L-1) L-1) (°C) Day 11 A NA NA 7,2 10 Day 11 B NA NA 7,68 10 Day 11 C NA NA 6,35 10 Day 11 D NA NA 6,73 10 Day 11 E 0,8 <0,05 7,43 10 Day 11 F NA NA 8,68 10 Day 11 G NA NA 6,99 10 Day 11 H 0,2 <0,05 6 10 Day 11 I 0,6 <0,05 7,9 10 Day 11 J 0,4 <0,05 7,77 10 Day 11 K 0,4 <0,05 7,67 10 Day 11 L NA NA 9,02 10 Day 11 M 0,6 <0,05 8,6 10 Day 11 N 0,8 <0,05 8,45 10 Day 11 O 0,025 <0,05 0,78 10 Day 11 P NA NA 8,37 10 Table VIII-8 Water quality parameters measured at the last day of the first screening test. N.A.: Not applicable.

Time Tank Nitrite (mg/L) Ammonium (mg/L) Oxygen (mg/L) Temperature (°C) Day 6 1a 0,2 <0,05 8,4 8 Day 6 1b 0,1 0,1 7,64 8 Day 6 1c 0,2 <0,05 7,99 8 Day 6 2a NA NA 7 8 Day 6 2b NA NA 8,05 8 Day 6 2c NA NA 7,05 8 Day 6 3a 0,1 <0,05 7,25 8 Day 6 3b 0,8 <0,05 8,14 8 Day 6 3c 0,2 0,1 5,2 8 Day 6 4a 1 <0,05 8,17 8 Day 6 4b 0,2 <0,05 8,06 8 Day 6 4c 0,05 0,1 5,05 8 Day 6 5a 0,2 <0,05 7,81 8 Day 6 5b 0,2 <0,05 6,7 8 Day 6 5c 0,4 <0,05 5,8 8 Table VIII-9 Water quality parameters measured at the day 6 of the first screening test. N.A.: Not applicable.

ANNEX 8 Isolated strains of which the DNA was extracted APPENDIX 93

Experiment Sampling Origin Colony Sample moment morpholog name y Mortality in larvae tank October larval tank yellow BactX water Mortality in stock Januari stock tank yellow 1A water Mortality in stock Februari stock tank green 1B water First adult screening test March LMG11229RIF yellow 2A tankwater First adult screening test April LMG11229RIF green 2B tankwater Effect of enriched environment and Januari Tank I yellow JanIa(1) physical stress on the host hemolymph Effect of enriched environment and Januari Tank I green JanIb(1) physical stress on the host hemolymph Effect of enriched environment and Januari Tank H, yelow JanHa(1 physical stress on the host hemolymph ) Effect of enriched environment and Januari Tank H, green JanHb(1 physical stress on the host hemolymph ) Effect of enriched environment and Januari Tank H, yellow JanHa(2 physical stress on the host hemolymph ) Effect of enriched environment and Januari Tank H, green JanHb(2 physical stress on the host hemolymph ) Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 3c 3c physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12

APPENDIX 94

Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c green 4c gr physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c yellow 4c g physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c yellow 4c g physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c yellow 4c g physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c yellow 4c g physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c yellow 4c g physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 2/02/2012 bak 4c yellow 4c g physical stress on the host hemolymfe 2/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 3/02/2012 bak 4c 4c physical stress on the host hemolymfe 3/2/12 Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat

APPENDIX 95

Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 6/02/2012 bak 3&4 6/2/12 physical stress on the host mosselhomoge naat Effect of enriched environment and 7/02/2012 bak 3&4 H10 physical stress on the host mosselhomoge naat Effect of enriched environment and 8/02/2012 bak 3&4 H11 physical stress on the host mosselhomoge naat Effect of enriched environment and 9/02/2012 bak 3&4 H12 physical stress on the host mosselhomoge naat Effect of enriched environment and 10/02/2012 bak 3&4 H13 physical stress on the host mosselhomoge naat Effect of enriched environment and 11/02/2012 bak 3&4 H14 physical stress on the host mosselhomoge naat Effect of enriched environment and 12/02/2012 bak 3&4 H15 physical stress on the host mosselhomoge naat Effect of enriched environment and 13/02/2012 bak 3&4 H16 physical stress on the host mosselhomoge naat

APPENDIX 96

Effect of enriched environment and 14/02/2012 bak 3&4 H17 physical stress on the host mosselhomoge naat Effect of enriched environment and 15/02/2012 bak 3&4 H18 physical stress on the host mosselhomoge naat Table VIII-10 Information of isolated strains for which the DNA was extracted for the ERIC PCR.

APPENDIX 97

Annex 9 Sequence alignment for Mytilin B and Lysozyme

CLUSTAL 2.1 multiple sequence alignment

…………………… gi|23263573|gb|AF334662.1| TTTCCTACGCCTTAAACCTTATATGCATGTTGTTATTACAAATAGGTTTT 1800 gb|DQ268868.1|_1-363 ------TTGCAGATGGG---- 82 ** ** ** ** gi|23263573|gb|AF334662.1| TTCATGTTAAAAGCTTTATGTTATTCCCCATCCTTTCTATAGGTAGAGTC 1850 gb|DQ268868.1|_1-363 ---ACGTTAA------89 * ***** gi|23263573|gb|AF334662.1| TCATTGCAACAACAATATAGGATGTCGTATGGATGTTGGTTCTTTGTCCT 1900 gb|DQ268868.1|_1-363 ---TTCGAAC------TCTT 100 ** *** ** * gi|23263573|gb|AF334662.1| GTGGACCCTTCCAGATTAAAAAGGCATATTGGATCGACTGTGGACAACCA 1950 gb|DQ268868.1|_1-363 GTGGATACATGCAAATAAAACAGGTTTACTGGGATGGCTGTGGAAAACCA 150 ***** * * ** ** *** *** ** *** * ******* ***** gi|23263573|gb|AF334662.1| AAAGGAGgtaacattattatttacaagaaaagaaatgcacacatgttgat 2000 gb|DQ268868.1|_1-363 GGCGGAAGT------TTAGAAG------CATGCT--- 172 *** ** *** *** **** * gi|23263573|gb|AF334662.1| gtagaaatgtacatcaatatatatgtattaattactattatctatatact 2050 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| tgcaatggtatgtgtttgaatttggatcctcaatgctcttcaacgtcgta 2100 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| ctttatttgacttttttgtcgatggtgtcagatgagtcttttaaagatca 2150 gb|DQ268868.1|_1-363 ------CCAAAGAT-A 181 ****** * gi|23263573|gb|AF334662.1| aacgcgcgtcttgcatttaaatttctaatccggatatctatgaatggagt 2200 gb|DQ268868.1|_1-363 AAC------184 ***

APPENDIX 98 gi|23263573|gb|AF334662.1| ttattttgaaccttctgtcctgcaaaattcaaaattcagtatgtcagtca 2250 gb|DQ268868.1|_1-363 --ATTGTG---CTTCT------CAGTGTGTC---CA 206 *** ** ***** **** **** ** gi|23263573|gb|AF334662.1| actggtagttgatacagttagcagtttctcatatagatttgagcaaggtt 2300 gb|DQ268868.1|_1-363 A------207 * gi|23263573|gb|AF334662.1| tttaccctcccaaatatatattgatgcaaaattgttttcagagaattttt 2350 gb|DQ268868.1|_1-363 ------AAATATATGTT------218 ******** ** gi|23263573|gb|AF334662.1| taaattgtgtgtatcatatattttgtgtaagtgctcatatactttgacca 2400 gb|DQ268868.1|_1-363 ------TAGGT------223 ** ** gi|23263573|gb|AF334662.1| aattgtacgagaaatagaattgatatgtcaaatgcgtaagcattggaatt 2450 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| tctcgatcaaatctattctgatttccgtatgtctgttgtatagggttatt 2500 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| ggtatgtaaaaagttgcatctgttatttaaacattaatcatacaccgtct 2550 gb|DQ268868.1|_1-363 ------ACATCAATCAT------234 **** ****** gi|23263573|gb|AF334662.1| ttggtttgtgcagatgactgaaacaagagaactcaaaattctaaaacgta 2600 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| agaagaaggttgaaaggatagcgagaaacgttacttattttgttgttgtg 2650 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| ttgcagtcttatagtaagacatgacacccaaaagtatctgtttaacaggt 2700 gb|DQ268868.1|_1-363 ------TATGG------239 *** * gi|23263573|gb|AF334662.1| aacgtgaaaacattttcaaaaaaatgaaattgaaagaatgggccatttac 2750 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| accactttatgtaatgcacaagtttcccagaagtttcacgtgatcacaat 2800 gb|DQ268868.1|_1-363 ------ATGCGCA------246 **** ** APPENDIX 99

gi|23263573|gb|AF334662.1| ataataaatgacatgacgatgacaatgattgtttatttcatttaagACTA 2850 gb|DQ268868.1|_1-363 ------CAT------249 *** gi|23263573|gb|AF334662.1| TAAGACATGTGCCAACGACTATTCATGTGCCTATAACTGCATCCAGACTT 2900 gb|DQ268868.1|_1-363 ------

gi|23263573|gb|AF334662.1| ATATGGCGAGGTACATTGGTCACAGTGGATGTCCTAAAAATTGTGAAAGC 2950 gb|DQ268868.1|_1-363 ------AATTGTGAGAGT 261 ******** ** gi|23263573|gb|AF334662.1| TATGCTCGAATCCACAATGGAGGTCCAAGGGGATGTACAAACCCAAACAC 3000 gb|DQ268868.1|_1-363 TATGCCCGAATGCCCAATGGAGGACCAGCAGGGTG------CAAACAC 303 ***** ***** * ********* *** ** ** ******* gi|23263573|gb|AF334662.1| CATTGGATATTGGAACAAGATCAAACAACAGGGTTGTACGATATATAGCT 3050 gb|DQ268868.1|_1-363 -ATTA-ATACT------TTGGGTTACGGGAGCCATATTC 334 *** *** * ***** ** *** gi|23263573|gb|AF334662.1| AAAGCCACCGGACATATAAATTAAATGTTCATGTTTAAACATAACAATTA 3100 gb|DQ268868.1|_1-363 AGAGCAA---GGGATGCA------GCGCA------AACAGTTA 362 * *** * * ** * * ** **** *** gi|23263573|gb|AF334662.1| AAAGACTTTTGAATTACTGGT 3121 gb|DQ268868.1|_1-363 A------363 Figure VIII-2 Relevant regions of lysozyme sequence alignment. gi|23263573|gb|AF334662.1|: Genomic DNA of M. edulis lysozyme; gb|DQ268868.1|_1-363 = M. edulis lysozyme mRNA, partial cds, used for primer design. Underlined sequences: coding DNA, small letters: intron

CLUSTAL 2.1 multiple sequence alignment

APPENDIX 100

……………………. gi|5815413|gb|AF177540.1| AAACTTAGTGCTGAATAAAATGACAAAATAGCAATTAACGAATTCCTATA 2150 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| TTTACTATTAACGCATTACAATTATTTTTATGTTTCAGTCCATGAGGCAG 2200 DNA|PRIMERDESIGN ------TCCATGAGGCAG 61 ************ gi|5815413|gb|AF177540.1| AGGCAAGTTGTGCTTCCAG- ATGTAAAGGCCATTGTAGAGCAAGACGCTG 2249 DNA|PRIMERDESIGN AGGCAAGTTGTGCTTCCAG- ATGTAAAGGCCATTGTAGAGCAAGACGCTG 110 ******************* ****************************** gi|5815413|gb|AF177540.1| TGGATATTATGTATCAGTC-- CTATACAGAGGGCGTTGCTACTGCAAATG 2297 DNA|PRIMERDESIGN TGGATATTATGTATCAGTC-- CTATACAGAGGGCGTTGCTACTGCAAATG 158 ******************* ***************************** gi|5815413|gb|AF177540.1| TCTTCGTTGTTCCAGTGAGCATTCCATGAAATTCCCTGAAAATGAAGGgt 2347 DNA|PRIMERDESIGN TCTTCGTTGTTCCAGTGAGCATTCCATGAAATTCCCTGAAAATGAAGG-- 206 ************************************************ gi|5815413|gb|AF177540.1| atgttgaacttgcaattgaaataatgatttgttttgtgtttttgtcttgc 2397 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| tcttgtatagtggtcaatatatttcttacataattggataggattttatg 2447 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| ttcaggttgatttcagatgttaattggctaacaacgtgtaacataaagag 2497 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| cataccgctaacatatacaagtgtaagtcgtttaaaaacatatgtagtgg 2547 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| attgagcacccgtaattagtccaaacgagtctaaatattataccgtacgc 2597 APPENDIX 101

DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| tgatttaaaaaaacaaaacatgaaagactcattttttttttataatctta 2647 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| cgttagataaaaacaattccgactttttaacatctcttctaaacatgcca 2697 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| tataataatatctatatctggatagcttaacttacaataaatttggttga 2747 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| attttttttcttaataacagtgaaagagatacttaataactgtcaaccaa 2797 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| ccttcaatcaacttcttaacataatgtaaaacacattttaagtattaatt 2847 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| ttgtttttgtttttagATCATCTCCATCTGACATGATGCCACAGATGAAT 2897 DNA|PRIMERDESIGN ------ATCATCTCCATCTGACATGATGCCACAGATGAAT 240 ********************************** gi|5815413|gb|AF177540.1| GAAAATGAGAACACTGAATTCGGTC- AGGACATGCCCACA-GGAGAAACC 2945 DNA|PRIMERDESIGN GAAAATGAGAACACTGAATTCGGTC- AGGACATGCCCACA-GGAGAAACC 288 ************************* ************** ********* gi|5815413|gb|AF177540.1| GAACAAGGTGAAACTGGCATTTAAAGAGATGATCCAATGATTCTCAGAAG 2995 DNA|PRIMERDESIGN GAACAAGGTGAAACTG------304 **************** gi|5815413|gb|AF177540.1| TGAAAATGACCCGTTCTGTTTGACATTATACAATTCTTCAATGTATTTTT 3045 DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| AATTGTTTAAGAGGTGTTTTTGATTTCTAAAATTGTAGCTCTTATCTGAA 3095 APPENDIX 102

DNA|PRIMERDESIGN ------

gi|5815413|gb|AF177540.1| TAAAAACTTAAATAGAAATGTGTATCGTTG 3125 DNA|PRIMERDESIGN Figure VIII-3 Relevant regions of sequence alligment for Mytilin B. gi|5815413|gb|AF177540.1|: Genomic DNA of M. galloprovincialis Mytilin B; DNA|PRIMERDESIGN: M. galloprovincialis mytilin B antimicrobial peptide precursor, mRNA, complete cds mRNA, partial cds used for primer design. Underlined sequences: coding DNA, small letters: intron

APPENDIX 103