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Diploma Thesis

Occurrence and distribution of the parasitic dinoflagellate Hematodinium sp. in decapod

crustaceans in Danish and Greenlandic waters

Submitted 30. January 2008 by

Falk Eigemann University of Rostock

Department of marine Biology Albert-Einstein-Str. 3

18059 Rostock Tel.: +49-(0)381/4986051 Fax : +49-(0)381/4986052

Supervisors:

Dr. Alf Skovgaard, University of Copenhagen

Dr. Stefan Forster, University of Rostock

Abstract ___________________________________________________________________________

Abstract This study focuses on the occurrence and distribution of the parasitic dinoflagellate

Hematodinium sp. that infects various decapod crustaceans. Three decapod crustaceans from

Danish waters (Nephrops norvegicus, Pagurus bernhardus and Liocarcinus depurator) and

two decapod crustaceans from Greenlandic waters (Chionoecetes opilio and Hyas araneus)

have been examined for infection.

All samples have been examined morphological by colour method (discolouration of

the carapace) and Nephrops norvegicus samples in addition by pleopod method (agglutination

of haemocytes and parasite cells in the pleopods). Further, DNA was extracted from eleven

Pagurus bernhardus, 72 Nephrops norvegicus, eight Liocarcinus depurator, 20 Hyas araneus

and 100 randomly selected Chionoecetes opilio samples and later tested by PCR for the

occurrence of a Hematodinium sp. infection. Primer sets used for detection were

Hematodinium-specific and amplified the ITS1 region and a small part of the 18 S rDNA.

Hematodinium sp. was detected in Nephrops norvegicus, Liocarcinus depurator and

Pagurus bernhardus from Danish waters and in Chionoecets opilio and Hyas araneus from

Greenlandic waters. All infections were detected by PCR whereas no infection could be

proved by colour or pleopod method.

The overall prevalence of infection for the respective hosts ranged between 40 and

87.5%, although sampling was done at periods of the year were infection rates are low. This

means that most animals dealt with a latent infection and indicates that the assumed general

deadly fate of an infection is not true.

The 27 obtained Hematodinium sp. ITS1 sequences from the five different hosts and

two different areas showed more than 98% similarity, except of two outlier sequences ex

Chionoecetes opilio. The two outlier sequences showed more than 83% similarity in the

variable ITS1 area, but revealed 6.7% difference in the partly sequenced conserved 18 S

rDNA to the remaining 25 Hematodinium sp. sequences. Interpretation of this result requires

further research. The remaining sequences should be classified as one Hematodinium species.

A phylogenetic tree was generated with my Hematodinium sp. ITS1 sequences and

Hematodinium ITS1 sequences from GenBank. The tree revealed that two different groups

exist in the genus Hematodinium which both warrant species status.

i

Declaration ___________________________________________________________________________

Declaration

Hiermit erkläre ich an Eides statt, dass ich die Arbeit mit dem Titel: „Occurrence and

distribution of the parasitic dinoflagellate Hematodinium sp. in decapod crustaceans in

Danish and Greenlandic waters“ selbstständig und nur unter Verwendung der

angegebenen Hilfsmittel verfasst habe.

__________________ ______________________

Ort, Datum Unterschrift des Verfassers

ii

Table of contents ___________________________________________________________________________

Table of contents

Abstract........................................................................... i

Declaration...................................................................... ii

Table of contents............................................................. iii

List of abbreviations....................................................... vi

List of figures.................................................................. vii

List of tables.................................................................... viii

Acknowledgements......................................................... ix

1. Introduction................................................................ 1

2. Background................................................................. 2

2.1. General aspects of dinoflagellates........................ 2

2.2. General aspects of parasitic dinoflagellates.......... 3

2.3. Taxonomic position of Hematodinium................. 4

2.4. Biology and ecology of Hematodinium................ 6

2.5. Hosts of the genus Hematodinium........................ 13

2.6. Effects on the host................................................ 19

2.7. Hematodinium effects on commercial

fisheries......................................................... ....... 22

iii


3. Material and methods................................................ 25

3.1. Methods for disease identification........................ 25

3.2. Sampling............................................................... 29

3.3. Colour and pleopod method................................. 30

3.4. DNA extraction.................................................... 31

3.5. PCR reactions....................................................... 31

3.6. Electrophoresis..................................................... 33

3.7. DNA purification.................................................. 34

3.8. DNA sequencing.................................................. 34

3.9. Sequence alignment.............................................. 34

3.10.Sequence comparison........................................... 34

3.11.Primer design........................................................ 35

3.12.Calculation of a phylogenetic tree........................ 35

4. Results.......................................................................... 36

4.1. Colour method...................................................... 36

4.2. Pleopod method.................................................... 37

4.3. PCR detection....................................................... 38

4.4. Sequence analyses................................................ 42

4.5. Phylogenetic tree.................................................. 43

5. Discussion.................................................................... 47

5.1. Summary of results............................................... 47

5.2. Proof of Hematodinium sp. in Danish waters....... 47

iv


5.3. Detection of Hematodinium sp. in Hyas

araneus................................................................. 48

5.4. Species discussion within Hematodinium............ 48

5.5. Prevalence of infection with Hematodinium........ 50

5.6. External reservoir of Hematodinium?................... 52

5.7. Latent infections with Hematodinium?.................53

6. Future aspects............................................................... 55

7. References..................................................................... 56

Appendix....................................................................... 65

v

List of abbreviations ___________________________________________________________________________

List of abbreviations Abbreviation Meaning

ATP adenosintriphosphat

bp basepair

DNA desoxyribonucleinacid

DOC dissolved organic carbon

dsDNA doublestranded DNA

Elisa enzyme linked immunosorbet assay

FAA free amino acid

g gram

GATC mix of bases G, A, T and C

Hz hertz

IFAT immunofluoreszent antibody technique

ITS1 first internal transcribed spacer

M molar

mbar millibar

min minute

ml milliliter

mM milli molar

ng nanogram

PCR polymerase chain reaction

ppt parts per thousand (salinity value)

rDNA ribosomal DNA

RFLP restriction fragment length polymorphism

rpm rounds per minute

S Svedberg unit (weight value)

SSU small sub unit

TEM transmission electron microscope

V volt

(w/v) weight/volumen percentage solution

µl microliter

µM micro molar

vi

List of Figures ___________________________________________________________________________

List of Figures: Figure 1: Basic anatomy of a thecate, dinokont dinoflagellate.......................................... 2

Figure 2: Life-cycle for Hematodinium.............................................................................. 7

Figure 3: Photo Nephrops norvegicus................................................................................ 14

Figure 4: Photo Chionoecetes opilio.................................................................................. 15

Figure 5: Photo Pagurus bernhardus................................................................................. 16

Figure 6: Photo Hyas araneus............................................................................................ 17

Figure 7: Photo Liocarcinus depurator.............................................................................. 17

Figure 8: Colour method, Chionoecetes opilio................................................................... 25

Figure 9: Colour method, Nephrops norvegicus................................................................ 26

Figure 10: Map of sampling stations................................................................................... 30

Figure 11: Map of primer binding sides.............................................................................. 32

Figure 12: Map of the rDNA............................................................................................... 33

Figure 13: Colour method, Chionoecetes opilio.................................................................. 36

Figure 14: Colour method, Nephrops norvegicus............................................................... 36

Figure 15: Pleopod method, Nephrops norvegicus............................................................. 37

Figure 16: Close-up view of Figure 15................................................................................ 37

Figure 17: Results for single PCR....................................................................................... 38

Figure 18: Results for semi-nested PCR............................................................................. 39

Figure 19: Results for nested PCR...................................................................................... 40

Figure 20: Prevalence of infection for Chionoecetes opilio................................................ 41

Figure 21: Gel of a nested PCR........................................................................................... 42

Figure 22: Phylogenetic tree of Hematodinium................................................................... 46

Figure 23: Comparison single PCR with nested PCR......................................................... 51

Figure 24: Gel of a single PCR............................................................................................ 51

Figure 25: Gel of a nested PCR........................................................................................... 51

vii

List of tables ___________________________________________________________________________

List of tables Table 1: Hosts of Hematodinium......................................................................................... 18

Table 2: Table of PCR approaches...................................................................................... 32

Table 3: Comparison fjord stations, offshore stations and stations on the edge................. 41

Table 4: Hematodinium sp. sequence numbers and respective host................................... 43

viii

Acknowledgements ___________________________________________________________________________

Acknowledgements This work would not have been possible without the encouragement and support of my two

supervisors, Dr. Alf Skovgaard (University of Copenhagen) and Dr. Stefan Forster

(University of Rostock). Especially Dr. Alf Skovgaard put much effort and research money in

success of this work.

Furthermore, I would like to express my gratitude to all people who have supported

me in many ways: All colleagues from the “Department of Phycology”, Copenhagen, who

made it a comfortable and progressive residence, especially Terje Berge for proof reading and

Anette Hørdum Løth for supporting and introducing me in the DNA lab; AnnDorte

Burmeister from the “Greenland Institute of Natural Resources” for organizing the research

cruise in Greenland; all people on board of the MS Adolf Jensen for making it an

unforgettable experience; Fisherman Paul Hansen from Gilleleje/Denmark for allocating

samples of Nephrops norvegicus and Liocarcinus depurator free of cost; the “Danish

Botanical Society” for funding the research tour to Greenland.

Special thanks to my son, Kurt Schadach, who offers me different views of life since

he is born and shows me the bright side of life every day we see us.

Last but not least I would like to thank my parents to enable my residence in Denmark and all

my friends who supported me during this time.

ix

Introduction ___________________________________________________________________________

1. Introduction Dinoflagellates represent the most diverse group of unicellular eukaryotic organisms

in terms of nutritional strategies (Taylor, 1987). This diversity is reflected in nomenclature

confusion because protozoologists viewed them as animals while phycologists viewed them

as plants. They were therefore placed into two different nomenclature systems as protozoa at

the one hand and algae at the other. Today, the term protists is used to include all groups of

unicellular eukaryotes. About 50% of dinoflagellates are raptorial predators that feed on other

protists, while the other half live entirely as plants (autotrophic). Some species even live both

as plants (autotrophic) and as animals (heterotrophic) simultaneously, making them

mixotrophic. A specialized way of dinoflagellate life is found among the approximately 140

species of parasitic forms. These parasites usually live osmotrophically outside or inside a

diverse array of different hosts. They normally kill their hosts, including both commercially

and ecologically important crustaceans and fish (Shields, 1994).

This thesis focuses on occurrence and distribution of one such parasitic dinoflagellate

genus in Greenland and Denmark, i.e. Hematodinium that infects different decapods.

Three decapod crustacean species from Danish waters, namely Pagurus bernhardus,

Liocarcinus depurator and Nephrops norvegicus and two decapod crustacean species from

Greenlandic waters, namely Chionoecetes opilio and Hyas araneus have been examined for

infection.

The purpose of this study is:

i) to prove the existence of Hematodinium in Danish waters

ii) to monitor the presence of Hematodinium in Chionoecetes opilio in Greenlandic

waters

iii) to compare Hematodinium DNA sequences from different hosts and different areas

to see if it is the same species or not

iv) to collect sequence data for outworking a reasonable phylogeny of the genus

Hematodinium

v) to compare morphological and molecular methods concerning the sensitivity of

detection for Hematodinium infections

1

Background ___________________________________________________________________________

2. Background 2.1. General aspects of dinoflagellates

Dinoflagellates are belonging to the Protists and are forming, together with Ciliata and

Apicomplexa, the clade Alveolata. All Alveolates possess alveoli (flat vacuoles) under the

pellicula (Gajadhar et al., 1991, Cavalier-Smith, 1993). Dinoflagellates combine certain

primitive characters of the prokaryots (continuously condensed chromosomes, low levels of

chromosomal basic proteins, low molecular weight of cytoplasmic ribosomal RNA) with

unusual eukaryotic features (high levels of repeated DNA, discrete phase of DNA synthesis,

presence of a spindle). Therefore, they were considered to occupy a position near to the base

of the eukaryotic evolutionary tree (Loeblich, 1976; Taylor, 1976, 1978, 1980) and to be

among the most primitive of eukaryotic groups (Loeblich, 1976; Taylor, 1980; Loeblich,

1984). However, recent phylogenetic studies including DNA-sequence analyses contradict

this hypothesis and place them as eukaryotic group that not occupies a position close to the

base of the eukaryotic evolutionary tree.

Figure 1: Basic anatomy of a thecate, dinokont dinoflagellate, ventral view. Picture by Evitt, 1985,

modified by Falk Eigemann

Most dinoflagellates possess two different flagella (Figure 1), one laterally directed

(transverse flagellum, 9+2 construction assisted by an axoneme, unique to dinoflagellates)

and the other beating posterior (longitudinal flagellum, 9+2 construction) (Taylor, 1987). The

transverse flagellum normally encircles the cell and is placed in a furrow called the girdle,

which separates the hypotheca from the epitheca (Figure 1). The longitudinal flagellum is

located in another furrow, which is placed ventrally, and called the sulcus. The organisation of

2

Background ___________________________________________________________________________

the girdle and sulcus varies a lot between species and can be used in some cases for species

identification. Dinoflagellates also possess modified vacuoles termed pusules with unknown

function. Normally there are two pusules per cell with opening canals to the flagellar bases. An important, distinct feature of dinoflagellates relates to the nuclear organisation, and their

unique nucleus is called a dinokaryon. The dinokaryon contains chromosomes, which do not

decondense during interphase, and contains very little basic protein. Nucleosomes are absent,

the nuclear envelope remains intact during mitosis and the spindle is extranuclear (Taylor,

1987). Every species includes at least one phase of their life cycle with a motile cell (a

mastigote) which possesses a single layer of flattened vesicles (alveolar vesicles) just beneath

the cell membrane. Either, these vesicles can contain cellulotic plates making up the

dinoflagellate armour (theca), or they can be empty. Thus, in naked (athecate) species thecal

plates are absent, while they are present in armoured (thecate) species (Taylor, 1987). The

mastigote stage is the dominating phase in most free-living species of dinoflagellates. It has

formed the basis for dinoflagellate taxomomy derived from morphology (morphospecies).

The dinoflagellate armour differs considerably among species and has been used to classify

both extinct and living species. In naked and in particular parasitic forms, the traditional

morphological features of the theca are hard to detect and they have proven more difficult to

discover and describe. However, recent advances in particular TEM and DNA techniques

have resulted in significant taxonomical rearrangements (e.g. Daugbjerg et al., 2000), and new

naked species of dinoflagellates are continuously described (e.g. De Salas et al., 2003). At

present, phylogenetic and taxonomical researchers of dinoflagellates, are increasingly more

relying on a combination of molecular (DNA sequences), biochemical (pigment signatures)

and morphological data (electron and light-microscopy) (e.g. Daugbjerg et al., 2000).

2.2. General aspects of parasitic dinoflagellates Until now, approximately 2000 species of dinoflagellates have been described,

whereof 140 are parasites (Drebes, 1984). Parasitic dinoflagellates were first discovered in

1906 by Chatton. This relatively late discovery is probably related to the fact that the majority

of them are hard to identify as dinoflagellates, because parasitism has created specialised

morphologies and physiologies. Especially intracellular parasites are hard to detect (Shields,

1994). Most of the common dinoflagellate features like pusules, sulcus, girdle, flagella and

cytopharyngeal funnel are difficult to detect in parasitic forms (Chatton, 1920; Cachon, 1964;

Chatton and Poisson, 1931; Cachon and Cachon, 1987; Shields, 1994), and parasitic forms

3

Background ___________________________________________________________________________

possess special morphological features not present in free-living forms (Cachon and Cachon,

1987). However, all parasitic dinoflagellates possess a free-living stage called dinospore,

which is thought to be responsible for the dispersal of the species (Cachon and Cachon, 1987).

In 1964, Cachon distinguished two categories of parasitic dinoflagellates: the

Blastodinida (subdivision Dinokaryota, 2.3.), which are essentially ectoparasites and the

Duboscquodinida (recent Syndinea, 2.3.), which are mostly intracellular parasites. Some

Blastodiniales affect Copepods where they are situated in the gut and the stomach, whereas

Duboscquodinidan (Syndinian) dinoflagellates affect several different kinds of invertebrates

and some genera, like Hematodinium, infect mainly decapods (Shields, 1994). These two

groups differ in the morphology of their vegetative phase, their nuclear development and the

structural and metabolic relations with their host (Cachon and Cachon, 1987). The

Blastodinida possess a theca whereas the Syndinea (former Duboscquodinida) are naked

(Cachon and Cachon, 1987). Cachon and Cachon confirmed with this arrangement the

polyphyletic origin of parasitic dinoflagellates presumed by Chatton.

Most parasitic dinoflagellates exhibit an exclusive heterotrophic nutrition (Cachon and

Cachon, 1987), but some species still possess chloroplasts. For instance, the trophocytes of

Blastodinium sp. supply approximately 50% of their energy budget trough photosynthesis

(Pasternak et al., 1984).

Parasitic dinoflagellates infect algae, protists, crustacean, annelids, cnidarians,

molluscs, salps, tunicats, rotifers, ascidians and fish (Chatton, 1920; Cachon, 1964; Lom,

1981; Cachon and Cachon, 1987; Shields, 1994; Coats, 1999). Within the crustaceans,

dinoflagellates infect copepods, amphipods, mysids, euphausiids and decapods (Shields,

1994).

2.3. Taxonomic position of Hematodinium In recent years, many discussions were preceded concerning phylogenetic

arrangements for dinoflagellates without finding a conclusive solution. In the present study, I

refer to the benchmark “A classification of living and fossil dinoflagellates” (Fensome et al.,

1993) which is still the most adopted concept.

Within the Dinoflagellata there are only two subdivisions: Dinokaryota and Syndinea.

The genus Hematodinium is placed in the Syndinea. The subdivision Syndinea is entirely

parasitic and comprises only one class, namely Syndiniophyceae that includes the order

Syndiniales. There are five families within the Syndiniales. The genus Hematodinium is

placed in the family Syndiniaceae (Fensome et al., 1993).

4

Background ___________________________________________________________________________

The approximately 25 species of parasitic dinoflagellates infecting crustaceans are

classified in two orders, the Blastodiniales (subdivision Dinokaryota) and the Syndiniales

(Shields, 1994). In the Syndiniales there are four genera parasiting crustaceans: Actinodinium

(copepods, doubtful status), Hematodinium (decapod crustacaen), Syndinium and

Trypanodinium (copepod eggs, doubtful status) (Shields, 1994).

The genus Hematodinium was first described in 1931 by Chatton and Poisson as

Hematodinium perezi from the hosts Carcinus maenas (Roscoff) and Liocarcinus depurator

(Luc-sur-mer) from the Bretagne respectively Normandy in France. So far, there is just one

other Hematodinium species described, namely Hematodinium australis. This species was

described in 1994 by Hudson and Shields from the host Portunus pelagicus in Australian

waters. Many researchers predicted that there are many more species within the genus

Hematodinium (e.g. Meyers et al., 1987; Stentiford and Shields, 2005), and presumed

geographical related as well as host-specific species. However, recent data (Hamilton, 2007;

Small et al., 2007b, c) contradict this by showing high sequence similarities in variable gene

parts of Hematodinium (ITS1 and ITS2) from several different hosts and areas. The

descriptions of Hematodinium perezi and Hematodinium australis are based exclusively on

morphological attributes. However, morphological observations are doubtful for the

phylogeny of parasitic dinoflagellates. For instance, the plasmodial stage of Hematodinium

from Callinectes sapidus, Carcinus maenas and Liocarcinus depurator is vermiform and

motile whereas the plasmodial stage from Chionoecetes bairdi, Portunus pelagicus and Scylla

serrata is round and immotile (Shields, 1994). Nevertheless, sequence analyses revealed that

Hematodinium infecting Callinectes sapidus, Scylla serrata and Liocarcinus depurator should

be grouped together (Small, 2007c) and Hematodinium infecting Carcinus maenas should be

classified to another group (Hamilton, 2007) (no Hematodinium sequences are available for

the other hosts).

Hematodinium infecting Callinectes sapidus was morphologically identified as the

type species Hematodinium perezi (Newman and Johnson, 1975; Couch and Martin, 1979).

However, species descriptions should be treated carefully, because recent data (Hamilton,

2007; Small et al., 2007b, c) contradict that the type species Hematodinium perezi described

from Liocarcinus depurator and Carcinus maenas by Chatton and Poisson is the same species

at all. Nevertheless, sequence analysis suggest that there exist at least two different groups of

Hematodinium that warrant species status. The first group infects Nephrops norvegicus,

Camcer pagurus, Carcinus maenas, Chionoecetes opilio and Pagurus bernhardus (Small et

al., 2007b; Hamilton, 2007) and the second group infects Callinectes sapidus, Portunus

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Background ___________________________________________________________________________

trituberculatus and Liocarcinus depurator (Small et al., 2007c). Within these groups sequence

similarity in the highly variable ITS1 region is more than 98% (Small et al., 2007b, c;

Hamilton, 2007). Since no names for these two different groups are existing and for most

other hosts no data concerning group belonging of the respective parasite are available, I will

following only use the genus name Hematodinium.

2.4. Biology and ecology of the genus Hematodinium Distribution:

The genus Hematodinium is cosmopolitan distributed and infects predominantly decapod

crustaceans (Hudson and Adlard, 1994). Epizootics caused by Hematodinium are known from

decapods from Alaska (Bower et al., 2003), the U.K. (Stentiford et al., 2002; Field et al.,

1992), the eastern United States (MacLean and Ruddell, 1978; Newman and Johnson, 1975;

Messick, 1994), Australia (Hudson and Shields, 1994), China (Xu, 2005 in: Small, 2007c),

Newfoundland (Taylor and Khan, 1995; Meyers et al., 1987; Pestal et al., 2003), France

(Wilhelm and Miahle, 1996) and Sweden (Taernlund, 2000).

Nutrition:

Hematodinium lacks chloroplasts and is completely heterotrophic (Shields, 1994). It lives in

the haemolymph or body cavities of its host, and obtains, like all Syndinidae, its energy and

nutrients by osmotrophy (Shields, 1994). It lives extracellular, unlike most other Syndinidae,

that live in the cytoplasm and sometimes even inside the nucleus (Cachon and Cachon, 1987).

Life cycle:

Unfortunately, no complete life cycle of Hematodinium as well as for any other syndinean

dinoflagellate is known. However, all recognized stages of dinoflagellates are haploid (except

the zygotes). Further, conjugation is only known for some species and no karyogamie has

been observed within the parasitic dinoflagellates.

The life cycles of Blastodiniales and Syndiniales include two phases: a vegetative

phase with a trophont and a reproductive phase with sporonts. The sporonts are believed to be

responsible for new infections and the resulting dinospores are biflagellated but losing their

flagella by contact with a new host. The dinospore might be swallowed passively with food or

attaches itself to the host by a posterior tentacle-like projection, which is homologous to a

peduncle (Shields, 1994). During the trophic phase in the host, parasitic species lose their

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Background ___________________________________________________________________________

dinoflagellate morphology, and girdle, sulcus and flagella disappear. Only basal bodies and

the amphiesma remain intact (Cachon and Cachon, 1987).

Appleton and Vickerman (1998) have been investigated the most complete lifecycle for

Hematodinium (ex Nephrops norvegicus) in an in vitro culture (Figure 2).

Figure 2: Life cycle for Hematodinium ex Nephrops norvegicus

The principal multiplicative form in vitro is the multinucleate filamentous trophont (1), which undergoes growth,

branching and fragmentation. In older cultures, multi-branched filaments form radiating gorgonlocks colonies

(2) which may undergo compaction to form more spherical clump colonies (3) or attach to the substratum and

become flattened arachnoid trophonts (4). The latter are capable of outward growth and fusion with one another.

The syncitial arachnoid becomes a sporont when it synthesizes trichocysts and generate masses of sporoblasts

from its raised centre (5). Detached multinucleate sporoblasts (6) may settle to become secondary arachnoid

sporonts (7) if introduced into fresh medium, otherwise they generate flagellated dinospores (8), either

microspores (9) or macrospores (10). Both types of spores germinate several weeks later, giving rise to a new

generation of filamentous trophonts. From: Appleton and Vickerman, 1998

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Background ___________________________________________________________________________

The sporoblasts generate macro- and microspores, which are both uninuclear (Fig. 2: 6, 9, 10).

When they are maintained in fresh medium (10% foetal calf serum in a balanced Nephrops

saline with antibiotics), they germinate and produce multinucleate unattached filamentous

trophonts after approximately five weeks (Fig. 2: 1). These trophonts multiply by

fragmentation and growth. If these filamentous trophonts are not subcultured, they give rise to

colonies of radiating filaments, called gorgonlocks (Fig. 2: 2). The gorgonlocks are attached

to the substratum and are forming arachnoid multinucleate trophonts (Fig. 2: 4), which later

become arachnoid sporonts (Fig. 2: 5). If the resulting sporoblasts are introduced to fresh

medium, they settle and become secondary arachnoid sporonts (Fig. 2: 7). Otherwise, they

synthesize trichocysts and flagella (Fig. 2: 8) and become dinospores that start a new cycle.

Eaton et al. (1991) developed a partial life cycle for Hematodinium ex Chionoecetes

bairdi and Shields and Squyars (2000) designed a partial life cycle for Hematodinium ex

Callinectes sapidus. In the latter, a dinospore either from the water column or from a benthic

organism enters the host and grows into a plasmodial stage (multinuclear), which developes

into the trophont. The trophont turns into a sporont, which produces dinospores that leave the

host and start a new cycle.

Anyway, there are other studies showing different life cycles for Hematodinium in

other hosts, and none of them is complete, but every life cycle is showing three different

phases:

1. a multinucleate plasmodial stage

2. a vegetative phase where a trophont is produced via merogony

3. an asexual reproductive phase where sporonts are produced via sporogony

(Stentiford and Shields, 2005)

The trophonts (vegetative cells) live in the haemolymph and proliferate rapidly via

schizogony. The trophont develops into a plasmodial stage that possesses two up to eight

nuclei. Motile plasmodial forms or trophonts described by Appleton and Vickermen (1998)

have only been observed in Carcinus maenas (Chatton and Poisson, 1931), Callinectes

sapidus (Newman and Johnson, 1975; Mesick, 1994; Shield and Squyars, 2000) and

Nephrops norvegicus (Field et al., 1992). Appleton and Vickerman (1998) suggested that the

motile form might be the early developing trophont phase of all species of Hematodinium.

The plasmodial stage can become a trophont again or can develop into a pre-spore. These pre-

spores abandon the host through small vacancies in the skeleton such as the antennal glands,

the gills and probably through other apertures (Shields, 1994). The dinospores possess two

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dissimilar, laterally inserted flagella (like typical mastigote dinoflagellate stages), whereof

one is undulating and the other is trailing. All Syndinea produce macro- and micro-spores

(Chatton, 1920; Meyers et al., 1987; Jepps, 1937; Coats, 1988; Eaton et al., 1991).

Hematodinium macrospores are up to 15 µm in length and the microspores are up to eight µm

in length. There is only one type of spore per host individual. It is unlikely that these micro-

and macrospores are gametes since they have approximately the same DNA concentration in

the nucleus as the trophont (Shields, 1994), and meiosis and gamogony have not been

detected for Hematodinium.

The spores can survive in sea water for several days (up to 73 days in sterile seawater,

Meyers et al., 1987) but their fate is unknown (Shields, 1994). Frischer et al. (2006) proved a

free-living stage of Hematodinium ex Callinectes sapidus. This was the first detection of

Hematodinium outside a metazoan host.

Duration of infection:

No clear data exist concerning the duration from infection of a host to development of the

disease, and the time requested for sporulation. In Chionoecetes opilio infections appeared to

take 9-12 months to develop into a disease (Shields et al., 2005), and in Callinectes sapidus

the disease needed 30-40 days to progress (Shields and Squyars, 2000). In Chionoecetes

bairdi sporulation of Hematodinium took place 9-18 months after infection (Shields, 1994).

Nevertheless, Shields (1994) suggested that the life cycle is probably much shorter. In the

Hematodinium ex Nephrops norvegicus culture, the time from isolation to sporogenesis was

5-155 days (between 20 and 30 days in the majority of cases). Germination occurred 18-62

days (average 35 days) after sporogenesis. All cultures were maintained at 6-10°C (Appleton

and Vickerman, 1998).

Mode of infection:

In addition, the mode of infection is unknown. There are several possibilities for its route:

Some, so far unknown, long-living resting stages of Hematodinium which are ingested with

the food, transmission via cannibalism (Sheppard et al., 2003), and reservoir hosts like benthic

amphipods (Johnson, 1986; Small et al., 2006) were suggested. The latter could transmit the

parasite when eaten.

Infections in Chionoecetes bairdi (Meyers et al., 1987), Callinectes sapidus (Shield

and Squyars, 2000) and Portunus pelagicus (Hudson and Shields, 1994) have been

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transmitted in vitro via inoculation of haemolymph from infected hosts. In inoculation

experiments, it was possible to infect hosts with filamentous trophonts, vegetative amoeboid

trophonts (Meyers et al., 1987; Hudson and Shields, 1994; Shields and Squyars, 2000) as well

as with micro- and macrospores (Eaton et al., 1991). The role of the dinospores is also not

clear yet. There are discussions if it is a true transmission stage or just an intermediate stage

preceding a resting cyst or another non-parasitic stage (Shields, 1994). Frischer et al. (2006)

proved for a free-living stage of Hematodinium infecting Callinectes sapidus the ability to act

as an infective agent. Therefore, he suggested a waterborne disease transmission.

Hydrological distribution:

Hematodinium epizootics are following several hydrographical features. It seems that there

are higher levels of infections close to land than in offshore areas (Taernlund, 2000), and

epizootics occur often in unique hydrological areas like fjords and poorly drained estuaries

(Shields, 1994). Meyers and Co-workers observed an infection rate up to 100% in shallow

areas with narrow water bodies for Chionoecets bairdi (Meyers et al., 1987). In Necora puber

and Cancer pagurus the outbreaks at the English Channel were associated with embayment or

shallow lagoons (Latrouite et al., 1988; Wilhelm and Miahle, 1996). In Callinectes sapidus,

Chionoecetes opilio and Nephrops norvegicus outbreaks were also associated with constricted

areas (Messick and Shields, 2000, Meyers et al., 1987, 1990; Eaton et al., 1991; Field et al.,

1992; Field et al., 1998; Stentiford et al., 2001b; Pestal et al., 2003; Shields et al., 2005).

However, some outbreaks are also known from more open areas (Meyers et al., 1996; Field et

al., 1998; Briggs and McAliskey, 2002; Stentiford et al., 2002). In these open ocean systems

(e.g. Chionoecets opilio and C. bairdi in the Bering Sea), the prevalence of Hematodinium

was variable with most but not all regions exhibiting low prevalence (Meyers et al., 1996).

There are also several conditions required for a continued epizootic of Hematodinium, such as

relatively closed host populations, low water exchange and stressful conditions (e.g. high

temperatures, seasonal hypoxia, seasonal fishing and predation pressure) for the host

population (Shields, 1994). The depth seems also to be an important factor. Chionoecetes

opilio females showed a two times higher prevalence of infection for areas deeper than 250 m

compared to shallow areas (Pestal et al., 2003), and infections are generally rare at depths less

than 200 m (Shields et al., 2005). Shields et al. (2005) observed also the type of substrate to

be important. The prevalence of Hematodinium infections was highest in crabs from muddy

or sandy habitats suggesting an alternate host or a dietary factor influencing the transmission.

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Limiting factors:

Low water temperatures and low salinity (Messick et al., 1999) limit the proliferation of

Hematodinium in the host haemolymph. The salinity needs to be greater than 12 ppt

(Shields et al., 2003) and probably regulates the distribution of the parasite (Shields, 1994).

Nephrops norvegicus from the Irish Sea showed a significant positive correlation between

prevalence of Hematodinium infections and salinity (Briggs and McAliskey, 2002). In

Callinectes sapidus, Hematodinium is restricted to crabs from high salinity waters of the mid-

Atlantic and Gulf States (Messick and Shields, 2000). In general, observations of

Hematodinium are rarely reported below 18 ppt (Newman and Johnson, 1975; Messick and

Sinderman, 1992; Messick and Shields, 2000), and almost all infections of Hematodinium

have been reported for stenohaline host species. Frischer et al. (2006) valued the correlation

between salinity and prevalence of Hematodinium infections as another hint for a free-living

stage and respectively for a waterborne disease. In crustacean haemolymph or tissues, only

little changes in salinity appear (Frischer et al., 2006) and hence they concluded that only a

free-living stage could be limited based on salinity.

In Callinectes sapidus the intensity of Hematodinium infections increased during

warmer temperature (above 15°C) and decreased at lower temperatures (less than 16°C)

(Messick et al., 1999). The in vitro cultures of Hematodinium ex Nephrops norvegicus

indicated that the life cycle proceeds above 8°C but is retarded when temperatures exceed

15°C (Appleton and Vickermen, 1998).

Nephrops norvegicus examined by Taernlund (2000) revealed a higher prevalence of

infection when trawled during the night compared to trawling during the day. Hematodinium

infections also showed high patchiness (Wilhelm and Boulo, 1988; Wilhelm and Miahle,

1996; Taylor and Khan, 1995). For instance, Callinectes sapidus revealed infection levels of

70-100% and 0.1-10% respectively in nearby areas (Shields et al., 2003).

Host factors affecting the infection rate:

Hematodinium infection rates are associated with several host factors, including size and age

(Field et al., 1992, 1998; Messick, 1994; Stentiford et al., 2001b), sex (Field et al., 1992;

Shields et al., 2003; Stentiford et al., 2001b) and moult conditions (Meyers et al., 1987, 1990;

Eaton et al., 1991; Field et al., 1992; Shields et al., 2005). In addition, crustaceans appear to

be particular vulnerable to infection during oviposition and sexual contact.

Newly moulted Chionoecetes opilio and C. bairdi showed a higher prevalence of

infection than not recently moulted crabs (Meyers et al., 1990; Eaton et al., 1991; Dawe,

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2002; Shields et al., 2005). Large, male crabs of Chionoecets opilio revealed a significant

lower prevalence of infection compared to females (Dawe, 2002; Pestal et al., 2003; Shields et

al., 2005). In addition, infections in Callinectes sapidus appeared significant more abundant in

juvenile than in adult hosts (Messick, 1994; Messick and Shields, 2000). In Nephrops

norvegicus the highest rate of infection occurred in small females (Field et al., 1992), and

juvenile and female crabs of Chionoecets opilio and C. bairdi showed higher prevalence of

infections compared to adult, respectively male crabs (Stentiford and Shields, 2005). Based on

the different rates of infection in males and females, Field et al. (1992) suggested a correlation

with the different moulting frequency between the sexes.

However, in other studies differences in host factors were not correlated to the

infection rate (Messick, 1994; Eaton et al., 1991; Meyers et al., 1987). In Necora puber for

instance, no correlations were examined between infection rates and host size (Wilhelm and

Boulo, 1988). In general, conclusions for different infection peaks in the sexes are hard to

assess. The sexes differ in behaviour, physiology and methodology (Taernlund, 2000), and

this aggravates reasonable comparisons. Field and Co-workers mentioned that conclusions

concerning correlation between size and rate of infection are also hard to achieve, but it might

be a criterion of cohorts (Field and Appleton, 1995). Anyway, many researchers do not have

clear results concerning infection levels and host size (Latrouite et al., 1988; Wilhelm and

Boulo, 1988; Eaton et al., 1991).

Seasonality:

Most Hematodinium infections exhibit strong seasonal peaks in prevalence, but the patterns

are not the same for each host system (Stentiford and Shields, 2005). The infection rate of

Nephrops norvegicus at the Clyde Sea in Scotland shows peaks in winter and spring (Field et

al., 1992, 1998; Stentiford et al., 2001a, b). During the peaks, the prevalence of infection can

reach 70% (Field et al., 1992). In Chionoecetes bairdi from south-eastern Alaska prevalence

of infection increases through spring and peaks in summer (Meyers et al., 1990; Eaton et al.,

1991; Love et al., 1993). It declines through autumn, falling to zero at late winter when

previously infected crabs die (Meyers et al., 1990; Eaton et al., 1991; Love et al., 1993). In

Callinectes sapidus Hematodinium infections have a strong peak during the autumn, followed

by a remarkable decline in winter and a moderate increase in the spring (Messick and Shields,

2000; Sheppard et al., 2003). Cancer pagurus shows only small seasonality of infection in

France, but several spring samples showed consistent peaks through several years (Latrouite

et al., 1988).

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Generally, in boreal host species there are peaks during summer (C. bairdi) or fall (C.

opilio), whereas in more temperate host species outbreaks occur primarily during the fall (C.

sapidus) or late winter and spring (Nephrops norvegicus and Cancer pagurus). One common

pattern emerges in every host/parasite system: A nadir occurs when infections are extremely

low or even undetectable in host populations (Stentiford and Shields, 2005). Thus, there is a

latency of infection or an external reservoir for the parasite (Stentiford and Shields, 2005).

Seasonality of infection rates might be associated with host moulting (Meyers et al., 1990,

1996; Eaton et al., 1991) and maturation (Messick, 1994).

Apart from seasonality, epizootic periodicity in form of long-term cycles exists.

Juvenile Callinectes sapidus at the seaside bays of Maryland and Virginia showed infection

rates of 70-100% in 1991/92 (Messick, 1994). In 1996/97, prevalence ranged between 10 and

40% in the same area (Messick and Shields, 2000). In Nephrops norvegicus the Scottish

fishery reported prevalence of infection up to 70% in the early 1990s, whereas in the late

1990s it peaked around 40% (Field et al., 1998; Stentiford et al., 2001b). For Chionoecetes

opilio overall prevalence of infection increased steadily from 0.037% to 4.25% over ten years

(Pestal et al., 2003), reaching 9% in males and 25% in females in 2000 during an epizootic in

Conception Bay (Shields et al., 2005).

2.5. Hosts of the genus Hematodinium Hematodinium is a host generalist (Stentiford and Shields, 2005). Infections occur in

decapods all over the world with majority of infections in brachyuran crabs.

Nephrops norvegicus

The Norway lobster Nephrops norvegicus (L.) belongs to the decapod crustaceans, and lives

in self-grubbed burrows between 40 and 800 m depth on soft sediment. Nephrops norvegicus

only gets out of its burrow during night for food intake (Køie et al., 2001). It predates on

worms, fish and other crustaceans. The overall length can reach 24 cm but most individuals

are smaller and females (up to 20 cm) are mostly smaller than males, but no apparent sex

dimorphism exists. Around 60000 tons are caught annually and sold as scampi (Italy),

langoustine (France) and Langustenschwänze or Kaiserhummer (Germany). The edible part is

the tail and not the claws as in most other decapods. The distribution ranges from Iceland and

Norway in the northeastern Atlantic Ocean through the North Sea as far as Portugal and

Morocco in the south. Infections with Hematodinium were first recognized in 1992 (Field et

al., 1992).

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Background ___________________________________________________________________________

Figure 3: Nephrops norvegicus, Photo by Falk Eigemann

Chionoecetes opilio

The snow crab Chionoecetes opilio (Fabricius, 1788) belongs to the brachyuran decapods.

The species displays distinct sexual dimorphism. The male crabs are much larger than the

females and the females have a round abdomen, whereas the males’ abdomen shows a four-

sided pyramid shape. Male crabs are divided in two morpho-types: The small-clawed, mostly

immature type that moults frequently, and the big-clawed type, which is mature and moults

seldom if ever. The carapace is almost as wide as long and can reach 17 cm in males and 10

cm in females in wide. The width (including legs) can reach 90 cm for males and 38 cm for

females. Chionoecetes opilio lives between 20 and 420 m depth on sandy or muddy substrate

and feeds mostly on benthic invertebrates. Its distribution ranges from the northwestern

Atlantic and north Pacific down to Japan and Korea. Important fisheries exist in Greenland

and Canada where crabs are caught with baited traps. Worldwide 115000 tons were caught in

2000 (www.fao.org). Hematodinium infections are known since 1990 (Taylor and Khan,

1995).

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Background ___________________________________________________________________________

Figure 4: Chionoecetes opilio, Photo by Falk Eigemann

Pagurus bernhardus

Pagurus bernhardus (L.) lives as a scavenger and predator and grazes additionally on

microorganisms from stones. It lives on rocky and sandy grounds between zero and 140 m

depth and reaches approximately 10 cm in size (Køie et al., 2001). The abdomen is soft and

not protected with a shell. Due to the need of protection, Pagurus bernhardus lives in old

gastropod shells, which are exchanged during its growth to find a suitable house. Therefore,

the abdomen is twisted and fit into the coils of the gastropod shell. The right claws are much

bigger than the left ones. The distribution ranges from Russia and Iceland at the North

Atlantic trough the North- and Baltic Sea to Portugal in the south. Hematodinium infections

are known from the U.K. (Small, 2006; Hamilton, 2007).

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Background ___________________________________________________________________________

. Figure 5: Pagurus bernhardus, Photo by www.hillewaert.be

Hyas araneus

The great spider crab Hyas araneus (L.) belongs to the brachyuran decapods and lives on hard

and sandy substrates between the tidal zone and 350 m depth (Køie et al., 2001). There exists

no sex dimorphism and it can reach 10 cm in length and 8 cm in width. Hyas araneus masks

itself with many epiphytes like Porifera, Bryozoa and Hydroids. It also actively cuts algae

with its claws and put them onto its carapace. The most common nutrition is starfishes. The

distribution ranges from Iceland, Svalbard and European Russia in the north up to the North

Sea (English Channel) and western Baltic-Sea in the south. It was also found in the Antarctic

Peninsula where it was the first known benthic invasive species. This study includes the first

observation of Hematodinium infections in Hyas araneus.

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http://www.hillewaert.be/

Background ___________________________________________________________________________

Figure 6: Hyas araneus, Photo by www.osl.gl.ca , modified by Falk Eigemann

Liocarcinus depurator

The harbour crab Liocarcinus depurator (L.) lives between the lower shore and sublitoral to

450 m depth on muddy sand and gravel as predator and scavenger (Køie et al., 2001). The

carapace is approximately 51 mm wide and 40 mm long. The most outstanding attribute is the

reconstructed fifth paraeopod, forming a swimming leg. It can be found from Norway to West

Africa including the Mediterranean. Liocarcinus depurator is the first host where a

Hematodinium infection was recognized (Chatton and Poisson, 1931, type species

Hematodinium perezi).

Figure 7: Liocarcinus depurator

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Background ___________________________________________________________________________

Other hosts

Following other hosts of Hematodinium are known:

Host species Author Year of detection

Callinectes sapidus Newman and Johnson 1975

Cancer irroratus MacLean and Ruddell 1978

Cancer borealis MacLean and Ruddell 1978

Chionoecetes bairdi Meyers et al. 1987

Portunus pelagicus Shields 1992

Scylla serrata Hudson and Lester 1994

Ovalipes oscellatus MacLean and Ruddell 1978

benthic amphipods Johnson 1986

Necora puber Wilhelm and Miahle 1996

Callinectes similis Messick and Shields 2000

Cancer pagurus Stentiford et al. 2002

Carcinus maenas Chatton and Poisson 1931

Chionoecetes tanneri Bower et al. 2003

Hexapanopeus angustifrons Messick and Shields 2000

Libinia emerginata Sheppard et al. 2003

Maja squinado Latrouite unpublished

Menippe mercenaria Sheppard et al. 2003

Neopanope sagi Messick and Shields 2000

Panopeus herbstii Messick and Shields 2000

Portunus latipes Chatton 1952

Trapezia coerulea Hudson et al. 1993

Trapezia areolata Hudson et al. 1993

Table 1: Hosts of Hematodinium

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Background ___________________________________________________________________________

2.6. Effects on the host In general, a Hematodinium infection ends with the death of the host. Infected hosts show

alteration in organs, tissues, haemolymph and hormonal function (Stentiford et al., 2000;

Shields et al., 2003; Stentiford et al., 2003).

Host defence:

The most important host defensive reactions are related to haemocytes. Functions of

haemocytes include wound repair, clotting, phagocytosis, nodulation and encapsulation of

foreign material, tanning of the cuticle, carbohydrate transport, glucose regulation,

haemocyanin synthesis and possibly osmotic regulation (e.g. Bauchau, 1981).

Some host species experienced decreased haemocyte numbers during infection with

Hematodinium (Shields, 1994; Field and Appleton, 1995; Shields and Squyars, 2000). The

haemocytes of Callinectes sapidus were destroyed by physical disruption as well as extra

cellular enzymatic degradation (Shields, 2003), and showed declines of 50 to 70%.

Haemocytopenia associated with severe Hematodinium infections likely hinders the normal

immune response of clotting, phagocytosis, encapsulation of foreign material, initiation of the

prophenoloxidase system and the production of other antibiotic factors (Smith and Söderhäll,

1986; Smith and Chrisholm, 1992). Therefore, a decline of haemocytes facilitates the

development of lethal secondary infections reported from hosts with Hematodinium infections

(Meyers et al., 1987; Field et al., 1992; Stentiford et al., 2003), or leads to the loss of clotting

ability with death ensuring due to loss of haemolymph (Shields et al., 2003).

Mature hosts appeared less prone to develop a Hematodinium infection

compared to their juvenile counterparts (Meyers et al., 1987; Messick, 1994). The reasons for

this are not known yet, but it might be correlated to moulting frequency. Some infected blue

crabs were immune in laboratory studies. They showed an increase of granulocytes and did

not show haemocytopenia, a loss of clotting ability or changes in morbidity (Shields and

Squyars, 2000).

Macroscopic signs:

Hematodinium can exhibit rapid logarithmic growth within a host (Shields and Squyars,

2000). In Nephrops norvegicus replacement of haemocytes with up to eight times their

number of Hematodinium cells (Appleton and Vickerman, 1998) is reported. This leads to a

colour change of the haemolymph into a milky-white appearance in heavy infected

individuals (Newman and Johnson, 1975; MacLean and Ruddell, 1978; Meyers et al., 1987;

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Background ___________________________________________________________________________

Field et al., 1992; Love et al., 1993; Hudson and Shields, 1994; Hudson and Adlard, 1994;

Messick, 1994; Shields, 1994; Field and Appleton, 1995; Taylor and Khan, 1995; Wilhelm

and Miahle, 1996; Taylor et al., 1996; Shields and Squyars, 2000; Stentiford et al., 2000).

In addition, some hosts (e.g. Nephrops norvegicus and Chionoecetes opilio) showed a

discoloration of the carapace in advanced stages of the disease (Meyers et al., 1987).

Biochemical changes:

In general, an infection leads to a significant alteration to the host’s haemolymph chemistry.

Changes in osmoregulation can result from shifts in plasmaproteins, amino acids and other

compounds, leading to osmotic collapse and likely contribute to the cause of death (Stentiford

et al., 1999). The copper levels in infected Nephrops norvegicus were 35% lower (Field et al.,

1992) and the oxygen carrying capacity of the haemocyanin was 43% lower (Taylor et al.,

1996). Taylor and Co-workers presumed that the hosts are dying due to a lack of intracellular

oxygen since the oxygen consumption is also much higher, caused by respiration of the

parasites (1996). Rittenburg et al. (1979) observed a decrease of ATP together with glycogen,

caused by the oxygen consumption. Furthermore, lipid and polysaccharid inclusions in

Hematodinium cells suggested active feeding at the expense of the host (Stentiford and

Shields, 2005). Probably therefore the infection caused an absence of reserve cells which led

to starvation in heavily infected Cancer pagurus.

Infected Callinectes sapidus males showed lower levels of serum proteins and

haemocyanin, but the females did not (Shields et al., 2003). In the same study, the acid

phosphatase activity in infected crabs was quite high, whereas the level in uninfected crabs

was below detection limit. High levels of acid phosphatase in Hematodinium probably inhibit

innate host defence like the superoxid mediated cell death (Shields et al., 2003).

Shields et al. (2003) observed a decrease of glycogen levels in the hepatopancreas

from infected Callinectes sapidus of 50% in females and even 70% in males. A rapid decline

of glycogen reserves leads to a severe metabolic drain due to pathogens. Glycogen is also a

precursor of chitin (Stevenson, 1985), and therefore a decline may affect especially infected

youngsters if moulting is not successfully (Shields et al., 2003).

Blue crabs probably die because of metabolic exhaustion (Shields et al., 2003). If

infected, the haemolymph glucose levels of the hosts decline rapidly due to the logarithmic

proliferation of the pathogens, coupled with their metabolic needs during rapid growth. In

extreme cases, glucose levels can reach zero (Stewart and Arie, 1973; Pauley et al., 1975;

Spindler-Barth, 1976). Because of low glucose levels, the host behaves lethargy. Again,

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starvation results out of this, because infected hosts cease feeding (Stewart and Arie, 1973;

Taylor et al., 1996). Furthermore, starvation can cause declines in serum proteins and

haemocyanin levels.

Meyers and Co-workers found in infected Chionoecetes bairdi a considerable amount

of a substance produced by the vegetative stages of Hematodinium and considered if this

substance is toxic to the host and/or if this substance is responsible for the bitter taste of

infected snow– and tanner crabs (Meyers et al., 1987).

Impacts to tissues and organs:

Much of the clinical disease in the host is caused by the non-motile vegetative parasite

morphology (Meyers et al., 1987). Hematodinium cells are in close association with, or even

attached to the basal lamina of the hepatopancreas (Stentiford et al., 2003). During patent

infections, the haemal arterioles of the hepatopancreas are grossly dilated and filled with large

numbers of parasitic cells (MacLean and Ruddell, 1978; Meyers et al., 1987; Hudson and

Shields, 1994; Field and Appleton, 1995; Wilhelm and Miahle, 1996; Stentiford et al., 2002).

In heavily infected animals, the hepatopancreatic tubules degenerate and parasites are often

found within the lumen of the tubules (Meyers et al., 1987; Field and Appleton, 1995;

Stentiford et al., 2002). This can proceed into a pressure-induced necrosis and consequently

the hepatopancreas cannot work normally. The hepatopancreas normally produces digestive

enzymes and facilitates the absorption and storage of nutrients. Due to the big amount of

parasitic cells, several organ tissues degenerate and respiratory dysfunction occurs, probably

related to the reduced copper concentrations (Shields, 1994). Hematodinium disrupts the gills

and other tissues directly (Meyers et al., 1987; Field et al., 1992; Hudson and Shields, 1994;

Messick, 1994). This happens when the vegetative stages of the parasite divide over months

in the host. The surviving crabs of this stage of the infection finally die when the vegetative

stages sporulate.

The gross appearance of muscle tissue alters during infection in terms of water

content, mechanical structure and texture (Meyers et al., 1987; Field et al., 1992; Messick,

1994; Wilhelm and Miahle, 1996). In addition, the connective tissue of the muscles shows a

decrease (Field and Appleton, 1995). In infected Cancer pagurus almost a complete

degeneration of the claw musculature took place (Stentiford et al., 2002). Further, Nephrops

norvegicus showed severe disorganisation in the z-line regions of the muscles (Stentiford et

al., 2000).

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Background ___________________________________________________________________________

Hematodinium possesses also the ability to infiltrate other tissues including cardiac

and skeletal muscle (Sheppard et al., 2003; Shields and Squyars, 2000; Hudson and Shields,

1994), gills, eye stalk and gut connective tissues (Field and Appleton, 1995; Meyers et al.,

1987).

Different impact to the sexes:

The different sexes of Callinectes sapidus showed a different pathophysiology during a

Hematodinium infection. Most measurements showed a more severe impact to male hosts, but

the mortality rate was not significantly different (Shields et al., 2003). Parasitic dinoflagellates

of copepods and amphipods are typically parasitic castrators (Shields, 1994), but this impact

was not observed in decapods infected with Hematodinium. Nevertheless, infected females of

Nephrops norvegicus did not develop mature gonads (Briggs and McAliskey, 2002).

Cause of death:

In general, the death probably occurs because of organ and/or respiratory dysfunction as well

as secondary infections with bacteria or ciliates (Meyers et al., 1987). Heavily infected

Nephrops norvegicus and Chionoecetes bairdi died within hours when sporulation took place

(Love et al., 1993; Stentiford et al., 2001a). Laboratory mortality rates ranged between 50 and

100% for Chionoecetes bairdi (Meyers et al., 1987; Love et al., 1993), C. opilio (Shields et

al., 2005) and Nephrops norvegicus (Field et al., 1992). Experimentally infected Callinectes

sapidus showed a mortality rate of 87% over 40 days (Shields and Squyars, 2000).

Overall, it should be said that no impacts to the human health are known, also if you eat

infected animals (Stentiford and Shields, 2005).

2.7. Hematodinium effects on commercial fisheries Hematodinium infections are known from six crustacaen hosts that are important subjects of

fishery, namely Chionoecetes opilio, Chionoecetes bairdi, Callinectus sapidus, Nephrops

norvegicus, Cancer pagururs and Necora puber. In these hosts, Hematodinium showed

seasonal prevalence up to 85% (Callinectes sapidus, Shields et al., 2003) at some specific

areas. In 2006, Frischer and Co-workers proved a free-living stage of Hematodinium that can

act as an infectious agent, and classified it as a harmful algae bloom (HAB) species.

Accordingly, Hematodinium can form cryptic blooms and has a dramatic but cryptic effect on

host populations (Meyers et al., 1987; Wilhelm and Miahle, 1996; Messick and Shields, 2000;

Stentiford et al., 2000, 2001a, b; Pestal et al., 2003; Shields et al., 2005).

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Background ___________________________________________________________________________

In Cancer pagurus, Hematodinium causes the “Pink Crab Disease” (PCD). The name

concerns to the pinkish appearance of the carapace from infected crabs. The most important

fisheries for Cancer pagurus are located in the U.K. and France. The crabs caught in U.K.

waters are mostly transported alive to other European countries. In 2000/2001, most crabs

died during transportation and showed a pinkish coloured carapace, caused by Hematodinium.

In general, infected crabs show weakness and lethargy and die when stressed (Shields et al.,

2003). In 1999, there were caught 27000 t in U.K. waters with a value of approximately 32

million £ (UK sea fisheries statistics, 1999 in: Stentiford et al., 2002). There are no

estimations for the loss caused by Hematodinium but presumably, it is a high value.

The Callinectus sapidus fishery is an important part of the fishery at the eastern

seaboard of the USA. Hematodinium epizootics occur from Delaware to Florida (Newman

and Johnson, 1975) and into the Gulf of Mexico (Messick and Shields, 2000). Since 1992, the

disease reached prevalence of 70-100% in crabs from coastal bays in Maryland and Virginia,

with lower prevalence, ranging from 0.1-10% in eastern parts of Chesapeak Bay (Messick,

1994; Messick and Shields, 2000). The annual harvests in Chesapeak Bay are between 80 and

100 million pounds (Johnson et al., 1998). Alone in Virginia the annual loss due to

Hematodinium is estimated to be 250000-500000 $ (Shields, unpublished data in: Stentiford

and Shields, 2005). Between 1998 and 2003, dramatic declines ranging from 9.7 to 51%

appeared in harvests compared to the previous ten years average (Georgia department of

natural resources, 2004 in: Frischer et al., 2006). Five-year observations suggested a causal

relationship between the current decline of blue crab population and the disease caused by

Hematodinium (Lee and Frischer, 2004).

In Chionoecetes opilio and C. bairdi Hematodinium causes the “Bitter Crab Disease”

(BCD). In 1987, BCD was first observed in Chionoecetes bairdi (Meyers et al., 1987) and

since it has been reported in increasing numbers of commercial catches (Taylor and Khan,

1995). The snow crab fishery in Newfoundland and Labrador got in 1999 earnings of 300

million Canadian $ (Pestal et al., 2003). The meat of infected crabs has a bitter taste and is not

marketable. One single infected crab can ruin the flavour of an entire batch, which may lead

to great economic loss to the fishery industry (Meyers et al., 1987; Taylor and Khan, 1995).

BCD is known from Greenland, Alaska and Canada (Meyers et al., 1987, 1990). Preliminary

data indicate that since 1990, the prevalence of Hematodinium in snow crabs from

Newfoundland has increased, but until now, no quantitative surveys of prevalence were made

(Taylor and Khan, 1995).

23

Background ___________________________________________________________________________

For the Nephrops norvegicus fishery in Scotland an annual loss between 2 and 4

million ₤ has been estimated (Field et al., 1992; Field and Appleton, 1995). The annual

harvests have a value of approximately 20 million ₤ (Shields, 1994). In infected Nephrops

norvegicus severe departures occur in the biochemical profiles of muscle and

hepatopancreatic tissues which makes the meat unmarketable (Stentiford et al., 1999, 2000).

In the French velvet crab Necora puber, catastrophic declines in the stocks due to

Hematodinium (Wilhelm and Boulo, 1988; Wilhelm and Miahle, 1996) were reported for the

English Channel.

Anyway, most of the financial loss has not been calculated and the real costs of

outbreaks of Hematodinium epizootics are hard to assess since dead hosts quickly become

undiagnosable. In addition, mortalities occur primarily in juveniles and females (Shields,

2003; Shields et al., 2005) which are often not marketable, but have great influence to the

stocks of the whole population.

However, the fisheries have possibilities to influence epizootics. The level of infection

differs in harvests from different fishery methods, probably caused by the different behaviour

of infected crabs. Necora puber in France showed a significant higher prevalence of

Hematodinium infections in trawled samples compared to trapped samples (Wilhelm and

Miahle, 1996). The same pattern was observed for Chionoecetes opilio (Pestal et al., 2003;

Shields et al., 2005). However, it should be noted that trawls have a lower minimum size of

retention (compared to traps), and prevalence tends to be higher in smaller crabs (Pestal et al.,

2003; Shields et al., 2005).

Moreover, fishing practises may help to spread the disease, such as culling or

disassembly of the catch at sea, re-baiting with infected animals and moving animals between

locations (Shields, 2003; Shields and Overstreet, 2004). The shipping of living animals to

distant markets accommodates an increased potential for the introduction of pathogenic agents

to new regions. This has been recognized in the past e.g. for the shrimp culture industry

(Flegel, 1997; Lightner and Redman, 1998). Since the highest infection levels occurred in

smaller animals, the fishery should also take care to keep balanced age populations, which

avert epizootics (Stentiford et al., 2001b). If sensitive shipboard diagnoses would exist, newly

infected crabs could be harvested prior to the development of patent infections and thereby

reduce the necessity to cull heavily infected crabs later in the season. The reduced

dissemination of infected carcasses would also follow (Meyers et al., 1987, 1990). However,

these are future aspects, because so far no good shipboard diagnoses exist.

24

Material and methods ___________________________________________________________________________

3. Material and methods 3.1. Methods for disease identification: Morphological as well as molecular methods are available for the detection of Hematodinium.

Morphological methods without any implements (e.g. colour method) vary between the

different hosts, because the host species alter morphologically different when they are

infected.

Colour method:

Infected snow crabs show a distinct red or pink discoloration on the carapace, which gives

them a “cooked” appearance. Infected crabs also have an opaque, solid white ventrum, a

listless or lethargic behaviour and milky, discoloured haemolymph (Meyers et al., 1990;

Taylor and Khan, 1995).

Photo credit: Dave Taylor, DFO

Figure 8: Chionoecetes opilio, animals at the right side should be classified as infected, on the left side as

healthy

Infected Nephrops norvegicus show the same red coloration on the claws, carapace and telson.

On the carapace in the vicinity of the heart the coloration can be especially strong (Taernlund,

2000). Trough the ventral abdomen it is possible to see that the haemolymph of infected

animals is milky white instead of normally bluish transparent. Heavily infected individuals of

Nephrops norvegicus have additional a chalky, cooked appearance.

25


Figure 9: Nephrops norvegicus, the right lobster should be classified as infected, the left as healthy, Photo

by Susanne Taernlund

The carapace of heavily infected Cancer pagurus shows a pinkish appearance, attributing to

the name of the disease: “Pink crab disease”.

In general, all macroscopic signs of the disease only appear when the infection is in

advanced stages. Consequently, many infected animals are wrongly classified as healthy.

Furthermore, for many hosts nothing is known about any alteration in morphology.

Pleopod method:

For Nephrops norvegicus and Callinectes sapidus the pleopod method can be employed to

detect a Hematodinium infection (Meyers at al., 1990; Eaton et al., 1991; Field et al., 1992;

Hudson and Shields, 1994; Messick, 1994; Wilhelm and Miahle, 1996). A pleopod is

removed and observed under an inverted microscope. In case of infection, agglutination of

haemocytes and parasite cells in the haemal space of the pleopod can be seen. Using the

pleopod method, an infection can be discovered in an earlier stage compared to the colour

method (Field et al., 1992). A classification system for the progress of the infection has been

developed (Field et al., 1992; Field and Appleton, 1995), staging the disease from stage 0

(healthy) to stage 4 (late stage infection). However, this staging of infection should be treated

carefully, since other studies show contradictory results (Taernlund, 2000). Anyway, for

skilled persons it is an applicable and fast method in the field since the only requirement is an

inverted microscope.

26


Histological methods:

For microscopic histological examinations, several methods are used ranging from

haemolymph wet smears to different staining practises, e.g. Leishman’s stain (Field and

Appleton, 1995). The typical dinokaryon type nucleus (containing five V-shaped

chromosomes) can be important for identification, as well as the mitochondria morphology,

the characteristic dinoflagellate amphiesma in the trophonts, or the presence of vermiform

plasmodial forms (up to five nuclei) (Field et al., 1992; Field and Appleton, 1995; Appleton

and Vickerman, 1998; Shields, 1994). The trophonts (vegetative cells) are single-, bi- or

multinuclear and approximately 10 µm in size. For beginners the wet smear microscopic

technique contains some problems, because the trophonts resemble immature haemolymph

cells and are the most frequently observed stage. To distinguish between host haemocytes and

trophonts the granularity and the oval/ellipsoidal appearance of the parasitic cells are suitable

(Shields, 1994). Motile, flagellated dinospores are rarely seen.

Biochemical detection:

Until now, only one method is available to detect Hematodinium attributed to biochemical

parameters. Stentiford and Co-workers developed a plasma free amino acid technique (FAA)

for the detection of Hematodinium in the host (Stentiford et al., 1999).

Immunological techniques:

An indirect immunofluorescent antibody technique (IFAT) with polyclonal antibodies derived

from rabbits was developed for cultured Hematodinium ex Nephrops norvegicus (Field and

Appleton, 1996; Appleton and Vickerman, 1998). This technique is able to show subpatent

infections in tissues as well as in haemolymph samples, but it is not significant more sensitive

compared to stained histological detection. The benefit of this technique is that it is also

suitable for tissues where the infection occurs earlier compared to patent haemolymph

infections. It was the first technique showing that infections occur all over the year and thus

doubt the assumption of external hosts as reservoirs. Another available immunological

method to prove Hematodinium is a Western Blot approach, designed by Stentiford et al.

(2001a). In 2002, Small and Co-workers designed an ELISA (enzyme linked immunosorbent

assay) for the detection of Hematodinium in host haemolymph (Small et al., 2002). The

detection limit was 50000 parasite cells per ml haemolymph. The sensitivity of this ELISA

reaction is four times greater compared to the Western Blot system. Furthermore, an ELISA

reaction can deal with much more samples and does not need as much time as the Western

27


Blot and IFAT methods. Another benefit of the technique is the very small amount of

haemolymph that is required. Accordingly, animals do not need to get seriously damaged

which can be useful if additional research with the same animals is planned.

Polymerase chain reaction (PCR):

DNA extraction can be followed by a PCR (polymerase chain reaction). All available

Hematodinium-specific primers bind to the small sub unit (SSU) of the rDNA or the ITS1

region. The SSU rDNA and attached ITS1 and ITS2 regions are suitable targets for

phylogenetic examinations, because they contain highly variable regions (ITS1, ITS2) as well

as conserved areas (18 S, 5.8 S). Furthermore, nuclear ribosomal DNA occurs tandem-like in

several thousand copies in the genome (Appels and Honeycutt, 1986). Sequences of the 18 S

can define higher level phylogenies and confirm genera belonging, whereas especially the

ITS1 is useful for phylogenetic studies of closely related organisms (Hillis and Dixon, 1991;

Coleman, 2003; Brown et al., 2004, Skovgaard et al., 2005).

Hudson and Adlard designed the first Hematodinium-specific primers in 1994. The

PCR product of this primer set produces a double band in the gel because it also binds to a

part of the host DNA. The host DNA fragment is much longer than the Hematodinium

fragment that it is not a problem for identifying Hematodinium. In 2002, a new primer set

(Hemat-F-1487, Hemat-R-1654) for Hematodinium detection was created, which lowered the

detection limit to one parasite cell per 300000 host haemocytes (Gruebl et al., 2002).

Stentiford et al. (2002) and Sheppard et al. (2003) designed other Hematodinium -specific

primer sets. None of these primer sets is species-specific, since all of them bind at the

moderately conserved 18 S (forward) and 5.8 S (backward) part of the SSU rDNA. Small et al.

(2006) developed a Hematodinium -specific primer set amplifying partly the first internal

transcribed spacer (ITS1) and flanking 3´region of the 18 S rDNA, namely 18SF2 and ITSR1.

It was made for detection of the Nephrops norvegicus infecting Hematodinium species. ITSR1

binds to the variable ITS1 region, and thus this primer pair is species-specific.

From the presented techniques, in the present study colour method, pleopod method

and PCR based diagnostic were adopted.

28


3.2. Sampling: Samples of Pagurus bernhardus were collected at 16. April 2007 at the Øresund close to the

island Ven. Animals were trawled between 30 and 35 m depth with MS Ofelia from the

Department of Biology, Helsingør, Denmark. Eleven crustaceans were chosen randomly and

stored alive in a refrigerator box filled with freezer packs, in seawater. After landing,

crustaceans were directly transported to the Department of Biology in Copenhagen for further

treatment. Two samples were taken out of every animal. Approximately 1 ml haemolymph

was removed with a 2 ml syringe and sterile 0.8* 40 mm needles out of the soft abdomen or

the axillary of the paraeopods and transferred into prepared 1.5 ml tubes containing 300 µl

2*CTAB (100 ml Tris HCl, 20 mM NaEDTA, 1.4 M NaCl, 2% (w/v)

hexadecyltrimethylammoniumbromide, 0.2% ß-mercaptoethanol). In addition a piece of heart

tissue was removed with sterile tweezers and scalpel and likewise transferred to prepared 1.5

ml tubes containing 300 µl 2*CTAB. All samples were frozen at -20°C until later DNA

extraction.

Samples of Nephrops norvegicus were received trough an agreement with a local

fisherman from Gilleleje/ Denmark. The Norway lobsters were caught in the Kattegat in the

night of 25. April (32 animals) and 30. May 2007 (40 animals). After landing in the early

morning samples were stored alive in a refrigerator box on ice and immediately transported to

the Department of Biology in Copenhagen. The catch of the 30. May 2007 comprised

additionally eight crabs of Liocarcinus depurator. All samples were treated exactly like

described for Pagurus bernhardus.

Samples of Chionoecetes opilio and Hyas araneus were collected during a research

cruise in the Sisimiut region at the west coast of Greenland (Figure 10) from 4. June 2007 to

22. June 2007 on MS Adolf Jensen from the Greenland Institute of Natural Resources. Crabs

were caught in traps baited with cephalopods, with ten traps in a line at each station.

Chionoecetes opilio samples (heart tissue and haemolymph) were taken from stations 1, 4, 7,

18, 21, 29, 32, 42, 48, 54, 60, 502 and 508 with 20 animals for each station and 15 animals at

station 10 (Appendix 1). Any station comprises approximately 80 up to 500 caught crabs and

all samples were chosen randomly. Molecular work (PCR based diagnostic) has been done for

stations 7, 21, 29, 42, 48, 46 and 49 (Figure 10, red arrows). Hyas araneus samples were

taken from stations 46 (18 animals) and 49 (two animals). Two samples were taken on board

out of every animal directly after capture like described above, except of syringe volume (10

ml). For every station depth (start depth, end depth, mean depth), temperature and position

were measured (Appendix 1). At three stations, temperature gauges got lost.

29


4644

49

48

7

2142

29

100 miles

Figure 10: Positions of trap stations at the Sisimiut area (West coast of Greenland) (not all stations shown, red arrows according stations examined with a molecular approach)

3.3. Colour and pleopod method: Photos were taken of every sampled Chionoecetes opilio (275 crabs) from the ventral as well

as the dorsal side to compare these results (colour method) later with the molecular approach.

In addition, scientists on board looked at every caught crab to see if obvious infected animals

are present. This colour method was done for approximately 14000 crabs (70 stations). All

Nephrops norvegicus samples (72) were photographed from the ventral and the dorsal side to

compare these results (colour method) with the PCR based diagnostic and the pleopod method.

For the pleopod assessment of Nephrops norvegicus the third pleopod at the right side from a

ventral view was removed from every animal (72) with tweezers and put on a microscope

slide. All pleopods were examined under an inverted microscope to check if agglutination

between haemocytes and parasite cells occurs. Photos were taken from every pleopod to

compare the results from the pleopod method with the molecular approach and the colour

method.

30


3.4. DNA extraction: Material was stored in 300 µl 2*CTAB at -20°C in 1.5 ml tubes. Extractions were made for

all samples of Nephrops norvegicus, Liocarcinus depurator, Hyas araneus and Pagurus

bernhardus and for all Chionoecetes opilio samples from stations 7, 21, 29, 42 and 48.

Samples were thawed in 65°C water bath. Following, one 3 mm metal beat was added to

every tissue sample and approximately 1 ml 0.1 mm silica beats were added to every

haemolymph sample. For cell destruction, all samples were mixed for 2* 1 min at 30 Hz in a

Retsch© TissueLyser. Afterwards, the tubes were incubated for 45 min at 65°C in a water

bath. After this, 300 µl chloroform were added and all tubes were vortexed. Tubes were

spinned in a centrifuge at 20000 g for 15 min at 20°C. The upper face (200 µl) was transferred

into new 1.5 ml tubes if it was clear. When the upper face was cloudy, the chloroform step

and spinning were repeated. To the 200 µl from the upper face, 400 µl –20°C isopropanol

were added, the tubes were inverted a few times and incubated at -20°C for minimum 1 hour

(sometimes overnight). After incubation, tubes were spinned at 20000 g for 15 min at 4°C.

The supernatant was discarded and the pellet washed with 300 µl -20°C 70% ethanol. Tubes

were spinned again for 15 min at 20000 g at 4°C and the supernatant was discarded. Finally,

samples were dried in an oven at 65°C for approximately 20 min and then resuspended in 50

µl EB-buffer. Extracts were stored at -20°C.

3.5. PCR reactions: PCR reactions were made for all extracted samples. For every PCR reaction a master-mix was

created including 5 µl TQ-buffer (0,67 M TrisHCl pH 8,5, MgCl2, 0,166 M NH4SO4, 0,1 M

2Mercaptoethanol), 5 µl TMA (C4H12NCl), 5 µl Forward primer, 5 µl Reverse primer, 8 µl

autoclaved water, 20 µl GATC mix and 0,1 µl Taq-polymerase for each sample. Primers were

used as 10 µM solutions. For single PCRs 2 µl template from the DNA extraction were used

and for nested or semi-nested PCR reactions between 0,2 and 1 µl template from the first PCR

product were used. PCR reactions were set up in a Biorad® MJ Research PTC-200 thermal

cycler. The reaction conditions were, with some exceptions, as follows: Denaturation for 1

min at 94°C, primer annealing for 1 min at 58°C and elongation for 2 min at 72°C. Single

PCRs consisted of 35 cycles, semi-nested and nested PCRs consisted out of 20 up to 30 cycles.

Before main cycle started, a previous denaturation step for 1 min at 94°C was implemented.

All reactions ended with a final extension step at 72°C for 6 min. Afterwards samples were

cooled down to 10°C until electrophoresis was conducted.

31


For detection of Hematodinium in the different decapod crustaceans three different

PCR approaches (single PCR, semi-nested PCR, nested PCR) were used (Table 2).

PCR approach Primer Sequence Author

Single PCR Backward: ITSR1

Forward: 18SF2

5’ GAA GGG AAG GGG AGA AGA AGC

5’ CAG TTT CTG GAA GTG GCA GCT G

Small et al., 2006

Small et al., 2006

Semi-nested

1. PCR

Backward: innominate

Forward: 18SF2

5’ CGC ATT TCG CTG CGT TCT TC


Hudson and Adlard, 1994

Small et al., 2006

Semi-nested

2. PCR

Backward: Hem3R

Forward: 18SF2

5’ TAA CCC GAG CCG AGG CAT TCA


Eigemann

Small et al., 2006

Nested

1. PCR

Backward: innominate

Forward: Hemat1487F

5’ CGC ATT TCG CTG CGT TCT TC

5’ CCT GGC TCG ATA GAG TTG

Hudson and Adlard, 1994

Gruebl et al., 2002

Nested

2. PCR

Backward: Hem3R

Forward: 18SF2

5’ TAA CCC GAG CCG AGG CAT TCA


Eigemann

Small et al., 2006

Table 2: PCR approaches with respective primer pairs and authors

The single PCR approach consisted of primer pair ITSR1/18SF2 (Small et al., 2006)

developed for the detection of Hematodinium in Nephrops norvegicus. ITSR1 binds in the

ITS1 area and is Hematodinium-specific. 18SF2 binds to the 3` ending area of the 18 S rDNA

(Figure 11 and 12). Hosts were staged as infected when a band of approximately

380 bp appeared after electrophoresis. 52 samples of Nephrops norvegicus, 11 samples of

Pagurus bernhardus and 40 samples of Chionoecetes opilio (station 42 and 48) were

examined with this primer pair.

Because no infection could be proved in samples for Chionoecetes opilio using primer

pair 18SF2/ITSR1 an own primer (Hem3R) was developed. Another reason for creating a new

primer was, that available primers for the Hematodinium detection showed disadvantages, e.g.

affection for pin structures, GC-content and base composition (especially ITSR1). To achieve

a higher sensitivity, the primer was developed for an area of the rDNA that admitted a nested

PCR setup if combined with already available primers.

EF065717_Hematodinium_perezi_clone

Hemat-F-1487

Hematodinium sp. ex Nephrops no

18S_F2

FE_47_10b

EukA

ITS_R1

HEM3R

HA1994R

Figure 11: Map of primer binding sites

32


The semi-nested PCR approach consisted of primer pair 18SF2/5' CGC ATT TCG CTG CGT

TCT TC (Hudson and Adlard, 1994, innominate, following named HA1994R) in the first

PCR, and 18SF2 and Hem3R in the second PCR. The primer Hem3R was developed using

helping tools (oligonucleotid properties calculator) to avoid pin-structures, create desired

melting point and a right GC content. It binds to the 5`area of the 5.8 S rRNA gene and,

thereby, amplifies the entire ITS1 region if used together with 18SF2 (Figure 11 and 12).

Especially for sequence analysis concerning phylogenetic examinations this exhibited an

advantage since the ITS1 region is highly variable. Primer HA1994R binds to the 5.8 S rDNA

(Figure 11 and 12). This PCR setup was used for all (20) Hyas araneus samples.

The nested PCR implementation consisted of primer pair Hemat1487F (Gruebl et al.,

2002)/HA1994R in the first PCR and 18SF2/Hem3R in the second PCR. The primer

Hemat1487F binds in the 18 S part of the SSU approximately 200 bp downstream to the

18SF2 primer (Figure 11 and 12). Eleven samples of Pagurus bernhardus, 20 samples of

Nephrops norvegicus, 8 samples of Liocarcinus depurator and 100 samples of Chionoecetes

opilio were examined with the nested PCR approach. During the studies, it obviously

appeared that this is the most sensitive approach for the Hematodinium detection.

Figure 12: Map of the rDNA

3.6. Electrophoresis: Gels were made with 1.5% agarose containing EtBr for DNA staining. 4 µl template were

mixed with 2 µl LB-buffer and filled in the wells of the gel. Electrophoresis conditions were

between 130 and 150 V for approximately 15 min. Afterwards the gels were photographed

with a Kodak® Edas 290 apparatus and edited with the Kodak® 1D computer program.

33


3.7. DNA-purification: PCR products with clean bands from the electrophoresis were chosen for DNA purification

for later sequencing. PCR products (46 µl) were transferred to a NucleoFast® 96 PCR plate,

and purified by filtration with a vacuum at -400 up to -600 mbar for 15 min. Into each well

100 µl nuclease free water were added and removed with a vacuum at -400 up to -600 mbar

for 15 min as additional washing step for the DNA. For recovering of purified PCR samples

50 µl nuclease free water were added and the plate was shaken for 20 min at 600 rpm.

Following 50 µl containing the resolved purified DNA were removed from the well into

0.5 ml tubes.

3.8. DNA-sequencing: The dsDNA content of the purified PCR products was measured with an Eppendorf®

BioPhotometer using 5 µl template and 45 µl autoclaved water in a 1 cm cuvette. Afterwards

the adequate amounts for 500 ng DNA were transferred to new 1.5 ml tubes and dried over

night at room temperature or in a 65°C oven. For every sample 2 µl 5 µM primer solutions

(forward and backward primer) for sequencing reactions were created and poured in a 1.5 ml

tube. Macrogen® / South Korea made all sequencing reactions (Sanger method).

3.9. Sequence alignment: All sequences were verified and edited, and the respective forward and backward sequences

pair wise aligned with the ChromasPro® computer program. The resulting consensus

sequences from the respective samples were aligned using the Bioedit® or ClustalX®

computer program (Appendix 2). Sequences that showed no doubts for all bases after manual

editing were used for calculation of a phylogenetic tree with computer program Mr.Bayes®.

3.10. Sequence comparison: Values for sequence similarities were achieved trough comparison of relative pair wise

sequence alignment. All sequence comparisons were executed with the Bioedit® computer

program. After pair wise alignment with ClustalW® the single ends were cut away and

similarity was calculated including introduced gaps as differences.

34


3.11. Primer design: The primer Hem3R was designed using known Hematodinium sequences from GenBank and

own sequences achieved with already available primers (HA1994R, 18SF2, ITSR1), using the

computer program “oligonucleotid properties calculator”

(www.basic.northwestern.edu/biotools/oligocalc.html#helpbasic). For achieving the sequence

of the whole ITS1 area, the primer was placed into the 5.8 S rRNA gene and used together

with known primers bind to the 18 S rRNA gene respectively.

3.12. Calculation of a phylogenetic tree: To 19 own sequences 53 chosen sequences (including Hematodinium sequences from every

known and available host) of Hematodinium from GenBank were added and aligned with the

computer program Bioedit® or ClustalX® (Appendix 3). Parts of the sequences belonging to

the 18 S or 5.8 S rDNA were cut away after alignment with known sequences of related

organisms (AF472555: Amoebophrya ; EF065717: Hematodinium perezi clone) from

GenBank. Accordingly, only ITS1 sequences were used for calculation. Tree calculation was

done with computer program Mr.Bayes v.3.1.2.®, using default parameters except for

generations (100000) and sample frequency (50). Burnin was set at 500 after checking for

stationarity by examinating the log-likelihood curves over generations. The consensus tree

(50% majority rule) was then constructed with Mr.Bayes®.

35

Results ___________________________________________________________________________

4. Results: 4.1. Colour method: None of the examined individuals of Chionoecetes opilio (approximately 14000) showed clear

signs of a Hematodinium infection. According to the colour method all crabs should have

been diagnosed as healthy. In addition, none of 72 Nephrops norvegicus were proved to be

infected with Hematodinium by this method.

Figure 13: Chionoecetes opilio, sample shows no morphological signs of infection, but an infection was

detected by PCR. Photo by Falk Eigemann

Figure 14: Nephrops norvegicus, sample shows no morphological signs of infection, but an infection was

detected by PCR. Photo by Falk Eigemann

36

Results ___________________________________________________________________________

4.2. Pleopod method: None of 72 Nephrops norvegicus samples was diagnosed as infected referring to the pleopod

method. No agglutinations of haemocytes and parasite cells could be detected at all.

Figure 15: Nephrops norvegicus pleopod. No agglutination can be seen, but an infection was detected for

the animal by PCR. Photo by Falk Eigemann

Figure 16: Close-up view of Figure 13. No agglutination can be seen. Photo by Falk Eigemann

37

Results ___________________________________________________________________________

4.3. PCR-Detection Hematodinium infections could, thus, only be proven by PCR. Occurrence of an infection was

detected with three different PCR approaches (3.5., Table 2). An animal was staged as

infected if either the haemolymph or/and the heart tissue sample showed a clear band in the

right length. No correlation between sample type and infection rate was observed. Infections

were later confirmed by sequencing of some, but not all, samples with an unambiguous band.

Results for single PCR (primer pair 18SF2/ITSR1):

5,77

27,27

00

5

10

15

20

25

30

Nephrops norvegicus 52 samples Pagurus bernhardus 11 samples Chionoecetes opilio 40 samples

%

Figure 17: Prevalence of infection in % for different hosts proved with primer pair 18SF2/ITSR1

Three out of 52 samples of Nephrops norvegicus were detected to be infected using primer

pair 18SF2/ITSR1. Two out of 32 animals from the catch at 25.April, and one out of 20

animals from the catch at 30. May. For three out of eleven Pagurus bernhardus an infection

was proved and no infection could be proved for 40 Chionoecetes opilio samples (station 42

and 48) (Figure 17). All three infections for Pagurus bernhardus and two out of three

infections for Nephrops norvegicus were confirmed via BLAST searches with the achieved

sequences. In several samples primer pair 18SF2/ITSR1 produced double bands.

38

Results ___________________________________________________________________________

Results for semi-nested PCR:

15

40

0

5

10

15

20

25

30

35

40

45

1. PCR 2. PCR

%

Figure 18: Prevalence of infection in % for Hyas araneus by semi-nested PCR

In the first PCR using primer pair 18SF2/HA1994R three out of 20 animals showed bands of

approximately 420 bp. Using these PCR products as templates and using the primer pair

18SF2/Hem3R (semi-nested PCR) eight crabs showed bands of approximately 400 bp (Figure

18). Four semi-nested PCR products with bands in the right length were sequenced after

purification and confirmed the Hematodinium infection.

39

Results ___________________________________________________________________________

Results for the Nested-PCR approach:

0 0 0 0

87,5

65

46

81,82

0

10

20

30

40

50

60

70

80

90

100

Liocarcinus depurator 8samples

Nephrops norvegicus 20samples

Chionoecetes opilio 100samples

Pagurus bernhardus 11samples

%

1. PCR: Hemat1487F/HA1994R2. PCR: 18SF2/Hem3R

Figure 19: Prevalence of infection in % for different hosts by nested PCR (1. and 2. PCR)

Eight samples (7 infected) of Liocarcinus depurator, 20 samples (13 infected) of Nephrops

norvegicus (not included in the 52 samples examined with single PCR), eleven samples (9

infected) of Pagurus bernhardus and 100 samples of Chionoecetes opilio have been examined

using primer pair Hemat1487F/HA1994R in the first PCR (no bands at all) and primer pair

18SF2/Hem3R in the second PCR (nested PCR approach, 3.5. Table 2). 40 crabs (station 42

and 48) included in the 100 samples of Chionoecetes opilio were examined earlier by single

PCR (3.5. Table 2), showing no bands at all (Figure 17). With the nested PCR approach seven

crabs from station 42 and two crabs from station 48 showed bands of approximately 400 bp

(Figure 20). Station 48 showed signs of a higher infection rate, but since only two bands were

clear and at the right position only two animals were staged as infected.

With the nested PCR approach nine out of eleven Pagurus bernhardus were classified

as infected (81.82%, Figure 19) with Hematodinium, whereas with the former single PCR

setup only three out of the same eleven samples (27.27%, Figure 17) showed bands in the

right length.

40

Results ___________________________________________________________________________

Prevalence of infection for Chionoecetes opilio:

40

60

85

35

10

0

10

20

30

40

50

60

70

80

90

Station 7 Station 21 Station 29 Station 42 Station 48

%

Figure 20: Prevalence of infection in % for Chionoecetes opilio from different stations by nested PCR

Prevalence of infection in Chionoecetes opilio ranged between 10 and 85% for different

stations by nested PCR (Figure 20). There was no observable correlation between

temperatures, depth and the prevalence of infection respectively (Appendix 1). However, all

stations containing samples that were examined by PCR were deep water stations (206 - 421

m). A comparison between infected males and females showed an overall prevalence of

infection of 49.44% for males (44 infected out of 89) and 18.18% for females (2 infected out

of 11). This data should be treated carefully since only eleven females were examined (no

statistical relevance) and six females derived from station 48 where overall prevalence of

infection was only 10%.

A clear correlation was seen by comparing stations in fjords with offshore stations

(Table 3). The overall prevalence of infection at the inner stations was (more than three times)

higher compared to the offshore stations. Station 7 fits this trend since it was situated at the

entrance of a fjord (3.2. Figure 10) and appeared an infection level (40%) between inner and

offshore stations.

Station 21 Station 29 Station 7 Station 42 Station 48 Average

inner 60 85 72.5

edge 40 40

offshore 35 10 22.5

Table 3: Prevalence of infection in % for inner stations, stations on the edge and offshore stations

41

Results ___________________________________________________________________________

4.4. Sequence analyses All in all, 35 PCR products that showed a bright band in the right length on a gel were

sequenced.

Figure 21: Gel of a nested PCR (samples of Pagurus bernhardus): 10 samples were classified as infected

(2a, 2b, 3b, 4b, 6a, 6b, 7a, 8a, 9a and 10a) whereof 3 (2a, 6a and 7a) were sequenced. a = haemolymph

sample, b = heart tissue sample, neg. = negative control

All 35 sequences confirmed that the amplified gene product was the ITS1 region (and an

additional small part of the 18 S and 5.8 S rDNA respectively) of Hematodinium.

Confirmation was achieved via BLAST searches in GenBank. Eight of these sequences still

showed several doubtful bases after manual editing, thus, only 27 of 35 sequences were

aligned (Appendix 2).

Two ITS1 sequences of Hematodinium ex Chionoecetes opilio (FE 43-16b and

FE 43-19b) showed only 83% similarity to all other sequences, whereas the remaining 25

ITS1 sequences showed a similarity of more than 98% to each other. These two outlier

sequences showed also more than 99% similarity to each other when compared pair wise. The

two outliers were not host or habitat related, since at the same station (station 42)

Hematodinium ex Chionoecetes opilio sequences were achieved that belonged to the main

group.

42

Results ___________________________________________________________________________

However, in the aligned 27 sequences two types could be recognized: Sequences FE

23-13; FE 43-4b; FE 47-1b; FE 47-3b; FE 47-10b and FE 47-17a revealed a polymorphism

(insertion of GGA GGA) from position 401 to 406 (Appendix 2), whereas all other sequences

(included outliers Fe 43-16b and FE 43-19b) did not have this insertion. These two types of

ITS1 sequences were not host or habitat related since both types of Hematodinium sequences

could be found in Chionoecetes opilio, even from the same sample station (station 42).

Furthermore, FE 24-1b exhibited a single A as insertion at position 347 and FE 51-6b showed

an insertion of a T at position 432 (Appendix 2).

Considering solely the partly sequenced conserved 18 S rDNA, the two outlier

sequences revealed seven differences in 104 bases compared to all other FE-sequences and

sequences taken from GenBank (Appendix 4). The chosen sequences from GenBank were

known to belong to a different group of Hematodinium and showed in the variable ITS1 area

only approximately 50% similarity to all FE-sequences (included outliers). However, in the

104 bases of the conserved 18 S rDNA these sequences from GenBank revealed only one

mismatch compared to the main group FE-sequences (Appendix 4).

Sequence number Host FE 23-HC-6a Pagurus bernhardus FE 23-13 Hyas araneus FE 24 Chionoecetes opilio station 21 FE 43 Chionoecetes opilio station 42 FE 44 Chionoecetes opilio station 7 FE 47 Chionoecetes opilio station 29 FE 51 Liocarcinus depurator FE 52 Nephrops norvegicus Table 4: Hematodinium sequence numbers (comply PCR reaction) with respective hosts

4.5. Phylogenetic tree: A phylogenetic tree was constructed by Bayesian analysis. Since some of the aligned

Hematodinium sequences showed 100% sequence similarity (Appendix 2), finally only 19

own (FE-) sequences were used to develop a phylogenetic tree of Hematodinium. 53

Hematodinium ITS1 sequences from GenBank were added. Accordingly, 72 ITS1 sequences

were used in the calculation (Appendix 3). Partly sequenced 18 S and 5.8 S rDNA was cut

away, that solely ITS1 sequences were used for calculation.

43

Results ___________________________________________________________________________

Phylogenetic distance can be seen due to horizontal length of the respective branches.

The numbers in front of the branches are PP, i.e. the percentage of trees exhibit the node

(Figure 22).

The tree confirmed that two closely related groups exist within the FE-sequences: The

main group FE-sequences and the FE-outlier sequences (FE 43-16b; FE 43-19b). A clearly

different group could be seen with Hematodinium sequences achieved from GenBank from

hosts Callinectes sapidus (Uncultured clone), Liocarcinus depurator (Hematodinium perezi

clone), Scylla serrata and Portunus trituberculatus. In the latter (following called perezi-)

group, slight host-specific differences could be seen (Figure 22).

Surprisingly, the own Hematodinium sequences ex Liocarcinus depurator (FE 51)

were not placed into the perezi-group. The tree confirmed sequence comparisons where FE 51

sequences showed more than 83% similarity (more than 93% when omitting the outliers) to

all other sequences (FE and Gen Bank) placed in the group from hosts Nephrops norvegicus,

Cancer pagurus, Chionoecetes opilio, Hyas araneus, Pagurus bernhardus, Munida rugosa

and Portunus prideaux (Figure 22, following called second group). Comparisons between FE

51 sequences (ex Liocarcinus depurator) and Hematodinium ex Liocarcinus depurator

sequences from GenBank (Hematodinium perezi clone) revealed only approximately 50%

sequence similarity in the ITS1 area.

In the second group, all FE-sequences except from FE 43-16b and FE 43-19b

branched together, confirming that only minor differences existing in these ITS1 sequences

(more than 98% similarity). In addition, most sequences taken out of GenBank from hosts

Nephrops norvegicus, Cancer pagurus, Chionoecetes opilio, Pagurus bernhardus and

Carcinus maenas branched together with the main FE-sequences (Figure 22). Three

sequences from Hematodinium ex Nephrops norvegicus (DQ084245, DQ084246 and

EU031969) did not branch with the main group (but also in the second group), but

phylogenetic distances were very small. The same can be concluded for two Hematodinium

sequences ex Cancer pagurus (EU096198, EU096196) and one Hematodinium sequence ex

Carcinus maenas (EU096220). Hematodinium sequences from Munida rugosa and Portunus

prideaux branched together and revealed slight host-specific distances to the main branch of

the second group as well as among each other (Figure 22).

FE 43-16b and FE 43-19b occupied a position between the two main groups but

exhibited a status much closer to the second group (Figure 22).

No clear correlation between geography and the two main Hematodinium groups

could be seen. Samples containing Hematodinium sequences from the perezi-group were

44

Results ___________________________________________________________________________

achieved from China (ex Scylla serrata and Portunus trituberculatus), the English Channel

(ex Liocarcinus depurator) and the East coast of the USA (ex Callinectes sapidus).

Hematodinium sequences belonging to the second group stem from Denmark (ex Pagurus

bernhardus and Nephrops norvegicus), Scotland (ex Nephrops norvegicus, Carcinus maenas,

Munida rugosa and Pagurus prideaux), the English Channel (ex Cancer pagurus), Ireland (ex

Cancer pagurus), Newfoundland (ex Chionoecetes opilio) and Greenland (ex Chionoecetes

opilio).

45

Results ___________________________________________________________________________

Figure 22: Phylogenetic tree of Hematodinium

46

Discussion ___________________________________________________________________________

5. Discussion: 5.1. Summary of results: In the present study Hematodinium was detected in five different decapod hosts. The overall

prevalence of infection was 46% for Chionoecetes opilio, 87.5% for Liocarcinus depurator,

65% for Nephrops norvegicus (20 samples with nested PCR), 81.82% for Pagurus

bernhardus and 40% for Hyas araneus (semi-nested PCR) (Figure 18 and 19). All infections

were proved with a molecular PCR technique whereas no infection could be proved via colour

or pleopod method. All 27 obtained complete ITS1 sequences (FE sequences) showed a

similarity of more than 83%. Actually, 25 ITS1 sequences showed a similarity of more than

98% to each other and only two sequences were outliers with 83% similarity to the other

sequences (Appendix 2). These two Hematodinium ex Chionoecetes opilio sequences

exhibited more than 99% similarity to each other respectively. A phylogenetic tree was

created using Hematodinium ITS1 sequences obtained in this study (FE-sequences) and

sequences from GenBank. The tree revealed two different groups of Hematodinium sequences

(Figure 22).

The questions that arise from the outline above are whether an external reservoir for

Hematodinium is required for the spread and transmission of the parasite and if the so far

presumed deadly fate of an infection can be true, dealing with infection rates between 40 and

87.5%. Another question to resolve is the taxonomical status of the two groups revealed by

the plylogenetic tree and the status of the two obtained outlier sequences.

5.2. Proof of Hematodinium sp. in Danish waters: This is the first study reporting Hematodinium in Danish waters. Hematodinium sp.

was detected in three decapod species, namely Liocarcinus depurator and Nephrops

norvegicus from the Kattegat and Pagurus bernhardus from the Øresund. This result was not

surprising, because Hematodinium was previously detected for the Swedish west coast in

Nephrops norvegicus (Taernlund, 2000, colour and pleopod method). Infected Pagurus

bernhardus were proved before for the English Channel (Small et al., 2006, PCR approach).

Liocarcinus depurator is known to be infected with the perezi-group of Hematodinium (Small

et al., 2007c) from the English Channel, which is probably a different species compared to the

second group of Hematodinium (infecting Nephrops norvegicus, Cancer pagurus, Pagurus

bernhardus and Chionoecetes opilio). My Hematodinium ex Liocarcinus depurator sequences

47

Discussion ___________________________________________________________________________

should surprisingly be classified to the second group. Further treatment of this point can be

found at “5.4. Species discussion within Hematodinium ”.

5.3. Detection of Hematodinium sp. in Hyas araneus: This is the first study detecting Hematodinium in Hyas araneus. Sampling of Hyas araneus

was achieved by accident in traps baited with cephalopods for snow crab fishery. Since Hyas

araneus lives in the same habitat as Chionoecetes opilio, for which infections are known since

1990 (Taylor and Khan, 1995), it is not surprising that also Hyas araneus is infected with

Hematodinium. In general, Hematodinium is presumed to be a host generalist (Stentiford and

Shields, 2005) and could probably be found in all decapod crustaceans in areas where

epizootics occur. Sequence comparisons with Hematodinium sp. sequences from other hosts

in this study did not show any host related sequence differences, supporting the classification

as a host generalist (Stentiford and Shields, 2005).

5.4. Species discussion within Hematodinium: The traditional criterion for defining a microorganism species is, like in higher animals and

plants, the morphology. However, defining a unicellular species only by morphological

parameters is proved to be of limited use. In unicellular eukaryotes it may often only reach

taxonomical “class” level (Logares, 2004), and does not permit to go beyond.

The descriptions of the two described species of Hematodinium (type species

Hematodinium perezi, Chatton and Poisson, 1931; Hematodinium australis, Hudson and

Shields, 1994) are based on exclusive morphological parameters. Since no complete life cycle

of Hematodinium is known and the known phases differ considerably in morphology,

exclusive morphological descriptions should not warrant species status. Until now, analyses

based on molecular sequencing revealed two different groups in the Hematodinium species

complex (Small et al., 2007c) that warrant species status. These two groups were confirmed in

the present study by the phylogenetic tree (Figure 22) and ITS1 sequence comparisons

(approximately 50% difference between the two groups).

The first species of Hematodinium infects Callinectes sapidus, Liocarcinus depurator,

Scylla serrata and Portunus trituberculatus (Figure 22). Sequences named “Hematodinium

perezi clone” derived from Liocarcinus depurator were added to GenBank by Small (2006).

In addition, Newman and Johnson (1975) and Couch and Martin (1979) identified

Hematodinium infecting Callinectes sapidus as the type species Hematodinium perezi

48

Discussion ___________________________________________________________________________

described by Chatton and Poisson (1931). Consequently, the first species should be named

Hematodinium perezi.

However, a redescription of Hematodinium perezi is required since the original

description was derived from parasites of two different hosts, namely Liocarcinus depurator

and Carcinus maenas. When comparing Hematodinium ITS1 sequences from these two hosts

(approximately 50% similarity) it seems unlikely that it is the same species of Hematodinium.

The second species of Hematodinium infects Nephrops norvegicus, Cancer pagurus,

Chionoecetes opilio, Pagurus bernhardus, Munida rugosa, Portunus prideaux and Carcinus

maenas (Figure 22). Accordingly, Hematodinium infecting Carcinus maenas should not be

named Hematodinium perezi like the type species description by Chatton and Poisson.

Previous studies (Small, 2006, 2007a, b, c; Hamilton 2007) revealed that the two

species of Hematodinium infect a defined array of hosts and thus are host related. However, in

the present study Hematodinium ITS1 sequences from Liocarcinus depurator (FE 51) needed

to be classified as the second species. The obtained ITS1 sequences exhibited more than 98%

similarity compared to other sequences belonging to the second species (FE and GenBank

sequences). This means that both Hematodinium species can infect Liocarcinus depurator.

The present study is the first report that both species of Hematodinium can be found in one

host species. Anyway, samples of Nephrops norvegicus and L. depurator derived from the

same area, and since Hematodinium is known as a host generalist (Stentiford and Shields,

2005), this finding is not unlikely. But, this finding again creates confusion concerning the

type species description of Hematodinium perezi by Chatton and Poisson (1931). It is

impossible to find out which species of Hematodinium was described and if it was the same

species at all.

Anyway, I suggest that the first species (so far presumed host array: Callinectes

sapidus, Liocarcinus depurator, Portunus trituberculatus and Scylla serrata) should be

named Hematodinium perezi. Re-naming would create confusion referring to former studies

(Newman and Johnson, 1975; Couch and Martin, 1979). In addition, sequence analyses can

refer to the GenBank sequences of “Hematodinium perezi clone” (Small, 2006). For the

second species a name is required.

In the partly sequenced (104 bases) conserved 18 S area, Hematodinium sequences

belonging to the perezi-group revealed only one mismatch compared to the second

Hematodinium species. Surprisingly, my outlier sequences (FE 43-16b; FE 43-19b) showed

seven different bases (in 104 bases) in the conserved 18 S area compared to both species

(Appendix 4). This result questions if my two outlier sequences are as closely related to the

49

Discussion ___________________________________________________________________________

second species of Hematodinium as shown by the phylogenetic tree, where only ITS1

sequences were used for calculation (83% similarity). I am not able to evaluate if the two

outlier sequences belong to another strain of the second Hematodinium species or an

independent species.

5.5. Prevalence of infection with Hematodinium: Comparison between morphological and molecular methods:

One of the aims of this study was to compare molecular with morphological methods

concerning the sensitivity of detection. It was already known that the colour method can be

only successfully conducted if the infection is in an advanced stage (Meyers et al., 1987).

Nevertheless, it was surprising that at the research cruise in Greenland no single Chionoecetes

opilio crab from approximately 14000 showed morphological signs of infection. This crab

cruise is an annual event and the years before there could always be seen at least some

infected crabs. Based on the low visible infection rate it is surprising that an overall

prevalence of 45% for snow crabs was found using nested PCR. Furthermore, the pleopod

method is thought to be more sensitive than the colour method. In the present study, the

prevalence of infection in Nephrops norvegicus was 65% using nested PCR (20 animals) as

detective tool. No infection could be proved for 72 animals (including the 20 examined with

nested PCR) using the pleopod method, suggesting that this method is also only applicable for

advanced infections.

The new developed primer Hem3R:

The new primer (Hem3R) developed in this study and used in the nested and semi-nested

PCR approach is much more sensitive for the detection of an infection than any known

diagnostic tool. Until now, primer pair 18SF2/ITSR1 (Small et al., 2006) was the most

sensitive detective tool (single PCR). Rates of infection using this PCR setup were 27.27% for

Pagurus bernhardus and 0% for Chionoecetes opilio (Figure 17). Samples of Pagurus

bernhardus and Chionoecetes opilio were examined additionally with the nested PCR setup,

revealing a prevalence of infection for Pagurus bernhardus of 81.82% (Figure 19 and 23) and

for Chionoecetes opilio of 22.5% (station 42 and 48) (Figure 20). This shows that the nested

PCR is at least three times more sensitive than using primer pair 18SF2/ITSR1 (Figure 23).

Samples of Nephrops norvegicus were not examined with both, single and nested PCR.

However, 52 samples treated with single PCR exhibited a prevalence of infection of 5.77%

(Figure 17), and 20 other samples treated with nested PCR revealed a rate of infection of 65%

50

Discussion ___________________________________________________________________________

(Figure 19). Sampling at 30.05.2007 gave 40 animals of which 20 were treated with single

PCR (5% infected) and 20 with nested PCR (65% infected), suggesting also a higher

sensitivity of nested PCR.

0

22,5

27,27

81,82

0

10

20

30

40

50

60

70

80

90

Primer pair 18SF2/ITSR1 Nested PCR

%

Chionoecetes opilioPagurus bernhardus

Figure 23: Comparison of primer pair 18SF2/ITSR1 with nested PCR

Figure 24: Single PCR (Primer pair 18SF2/ITSR) Figure 25: Nested PCR Gel with P. bernhardus samples. Infection Gel with P. bernhardus samples. Infection detected in three animals (2, 6, 7) detected in eight animals (2, 3, 4, 6, 7, 8, 9, 10) Figure 24 and 25: a = haemolymph sample, b = heart tissue sample, neg. = negative control

51

Discussion ___________________________________________________________________________

Comparisons with previous studies:

Previous studies measured much lower rates of infection for the respective hosts. Snow crabs,

for instance, showed an overall prevalence of infection between 0.11 and 4.25% (4.25%

Pestal et al., 2003, using wet smears for detection; Newfoundland: 0.11%, Conception Bay:

3.7% Taylor and Khan, 1995, using the colour method for detection). Recently, infection

levels for decapod crustaceans from the Clyde Sea/Scotland were published revealing overall

prevalence of infection for Liocarcinus depurator at 13% and for Pagurus bernhardus at 19%

(Shaw et al., 2007). These results were achieved via PCR detection using either primer pair

DinoF (Kim et al., 2004 in: Shaw et al., 2007)/ITSR1 (Small et al., 2006) or primer pair

Hemat1487F (Gruebl et al., 2002)/ITS4 (White et al., 1990). In 2002, Briggs and McAliskey

published a study where infection rates in Nephrops norvegicus from the western Irish Sea

peaked by 18% (colour method). Neil et al. (2007) reported prevalence of infection for

Nephrops norvegicus in the Clyde Sea area in Scotland with peaks at 38% for females and

22% for males, using an immunoassay as detective tool.

Furthermore, many studies showed a higher rate of infection in juvenile animals

(Messick, 1994; Messick and Shields, 2000; Field et al., 1992), whereas all samples used in

the present study were adult crustaceans. In addition, Chionoecetes opilio samples were

achieved with baited traps and further studies exhibited more infected crabs in trawled

samples compared to trapped samples (Pestal et al., 2003; Shields et al., 2005). Consequently,

my results probably underestimate the actual infection level, but in spite of this allegorize the

highest known values for the respective hosts.

5.6. External reservoir of Hematodinium? The life cycle of Hematodinium is widely unknown and in the past an external reservoir was

proposed (Small et al., 2006; Johnson, 1986). This assumption mainly based on the fact that

periods existed during the year where no Hematodinium infections could be detected in

decapods. The conclusion was therefore that an external reservoir is needed to guarantee the

spread and transmission of the parasite. Small and Co-workers (Small et al., 2006) and

Johnson (1986) detected Hematodinium in amphipods and suggested that this might be the

searched external reservoir. Transmission was thought to proceed by feeding on these benthic

amphipods. However, these results should be considered carefully since no other researcher

was able to detect Hematodinium in amphipods.

In the present study sampling was conducted at periods of the year were the rate of

infection is low for the respective host (Taernlund, 2000; Pestal et al., 2003) and therefore

52

Discussion ___________________________________________________________________________

could not be detected with morphological methods. Nevertheless, as mentioned before,

prevalence of infection ranged between 46 and 87.5% (Figure 19) for the different hosts,

proved by nested PCR.

This study shows that an intermediate host is not required to maintain populations of

Hematodinium and it is unlikely that such external reservoir exists. This point is strengthened

by the proof of a free-living stage in the life cycle of Hematodinium which can act as an

infectious agent (Frischer et al., 2006), and enables the spread of the disease directly from

host to host.

5.7. Latent infections with Hematodinium?

By means of this study, Hematodinium is found to be much more common than previously

believed. So far, many studies concluded that a Hematodinium infection is deadly to its host

(e.g. Shields, 1994; Taernlund, 2000) and no records of disease recovery are known (Meyers

et al., 1987; Field et al., 1992, 1995). There is only one study indicating that a host could be

immune against the parasite (Shields and Squyars, 2000). In that study, immune crabs of

Callinectes sapidus exhibited an increase in granulocytes and were not developing

haemocytopenia, a loss of clotting ability or changes in morbidity.

Concerning the high prevalence of infection in the present study there are two possible

conclusions: First, the parasitic dinoflagellate Hematodinium has much less impact to its host

as thought. This would mean most of the animals were indeed infected but dealt with a latent

infection. Second, if the deadly fate of an infection would be true, all studies concerning the

life cycle of Hematodinium would have calculated much too short time for it. But, the second

point is refuted by many in vitro studies concerning the life cycle of Hematodinium and in

addition by in vitro studies where healthy hosts were inoculated and infected with

Hematodinium (e.g. Appleton and Vickerman, 1998; Frischer et al., 2006). These studies

revealed durations between days and months for completion of the life cycle respectively

outbreaks of the disease after infection.

I propose that the first conclusion is true. The predicted impact of Hematodinium can

not be true and I suggest that most crustaceans offer a latent infection which only breaks out

and becomes deadly if the host is stressed or in otherwise bad conditions. Widespread latent

infections are known from other alveolate parasites, such as Toxoplasma gondii (Jakubek,

2007) and therefore supporting this point.

If the former conclusions concerning the general deadly fate for the host were true,

probably most of the respective populations would have disappeared. With a few exceptions,

53

Discussion ___________________________________________________________________________

the fisheries of Nephrops norvegicus, Cancer pagurus, Callinectes sapidus, Necora puber,

Chionoecetes opilio and Chionoecetes bairdi are successful and not dealing with serious

problems due to Hematodinium. My samples of Nephrops norvegicus and Chionoecetes opilio

were caught during fishing cruises and no problems caused by Hematodinium are known for

fisheries in these areas. A bigger loss for the Callinectes sapidus fishery was reported for

Maryland and Virginia and observations suggested a relationship between declines in harvests

and the disease caused by Hematodinium. In fact it was never proved that Hematodinium is

the real agent for the decline and there might have been other conditions or infections with

harmful agents that accelerated the breakout of latent Hematodinium infections.

54

Future aspects ___________________________________________________________________________

6. Future aspects I propose that Hematodinium is not as serious for the fishery of several decapod crustacaens

as assumed in most former studies. Due to the patchy distribution of Hematodinium there

might be regional losses but I do not think that Hematodinium has a great impact to the

fishery in general. However, culling animals while sailing and baiting with crustaceans from

regions where outbreaks are known should be prevented.

The phylogenetic tree and sequence analysis revealed that in the genus Hematodinium

two groups are existent that warrant species status. I suggested naming the first group (so far

presumed host array: Callinectes sapidus, Portunus trituberculatus and Scylla serrata)

Hematodinium perezi. For the second group a name and a suggestive description is

necessitated. Species descriptions for protists should be based on morphological as well as on

DNA sequence attributes. Unfortunately I was not able to execute any morphological

observations on Hematodinium, caused by the absence of positive samples by colour and

pleopod method. However, since the ITS1 area is proven to be a suitable target for

phylogenetic studies of closely related organisms (Hillis and Dixon, 1991; Coleman, 2003;

Brown et al., 2004; Skovgaard et al., 2005), I can not see any reason to doubt the clear results

obtained from the phylogenetic tree. Anyway, because morphological observations of

Hematodinium are hard to interpret (unknown life cycle) other parts of the DNA (whole 18S,

LSU) should be consulted additionally for final species descriptions.

Sequence comparisons only of the partly sequenced conserved 18 S rDNA revealed a

much closer relation between the two main groups of Hematodinium (perezi and innominate)

than any of these to the obtained outlier sequences. Therefore, comparisons of the 18 S rDNA

contradict the result from the phylogenetic tree. Due to insufficient time it was not possible to

generate the whole 18 S sequence for the two outlier sequences, but probably more

differences would appear. Since three out of four primers were Hematodinium-specific

(nested PCR) it is unlikely that the outlier sequences belong to another group of organisms.

However, I am not able to evaluate the taxonomical status of the outlier sequences. Until now,

only very few 18 S sequences of Hematodinium are available in GenBank for comparisons. In

the future a complete 18 S sequence for my outliers and more 18 S sequences of

Hematodinium perezi as well as Hematodinium sp. should be submitted to GenBank, to be

able to accomplish continuative studies concerning the phylogeny of the genus Hematodinium.

55

References ___________________________________________________________________________

7. References Appels R. and R. L. Honeycutt. 1986. rDNA: Evolution over a billion years. In: Dutta SK (ed) DNA systematics, vol 2. CRC Press, Boca Raton, p 81-135 Appleton, P. L. and K. Vickerman. 1998. In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp.) associated with mortality of the

Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116:115-130 Bauchau, A. G. 1981. Crustaceans. In: Ratcliffe NA Rowley AF (eds) Invertebrate blood cells, Vol 2, arthropods to urochordates, invertebrates and vertebrates compared.

Academic Press, London, p 386-420 Bower S. M., G. M. Meyer, A. Phillips, G. Workman and D. Clark. 2003. New host and range

extension of bitter crab syndrome in Chionoecetes spp. caused by Hematodinium sp. Bull Euro Assoc Fish Pathol 23: 86-91

Briggs, R. P. and M. McAliskey. 2002. The prevalence of Hematodinium in Nephrops norvegicus from the Western Irish Sea. J. Mar. Biol. Ass. U. K. 82: 427-433 Brown, G., K. L. Hudson and K. S. Reece. 2004. Genetic variation at the ITS and ATAN loci

among and within cultured isolates of Perkinsus marinus. J Eukaryot Microbiol 51: 312-320

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64

Appendix 1

Temperature, depth, position (n = north, w = west, 66312850=66° 312,850`) and date for sampled stations Station Temperature

°CMean Depht

mPosition

n1Position

w1Position

n2Position

w2 Date

1 2,6 192 66312850 53042100 66313050 53046910 07.06.20072 -0,6 356 66322650 53060710 66320500 53060050 07.06.20073 -0,5 427 66313090 53135740 66312300 53130530 07.06.20074 -0,5 470 66318430 5316268 66317550 53150370 07.06.20075 -0,4 345,5 66326510 53205310 66325690 53201670 07.06.20076 -0,5 330 66328760 53241460 66328700 53236400 07.06.20077 -0,5 259 66331730 53321930 66332030 53317680 08.06.20078 0,9 198,5 66312060 53414180 66314320 53413290 08.06.20079 1,1 174 66310910 53552240 66308850 53552980 14.06.2007

10 1,0 152 66339410 53530680 66337550 53529790 14.06.200711 1,4 213,5 66380812 53598060 66383140 53596800 14.06.200712 1,1 192,5 66362800 53591340 66364090 53588580 14.06.200717 -0,2 235,5 66414340 53216810 66414990 53212090 08.06.200718 -0,8 228 66425180 53189290 66425740 53164550 08.06.200719 -1,0 229 66421270 53119610 66420130 53115560 08.06.200720 -0,8 180,5 66424430 53072020 66424390 53065580 08.06.200721 -1,0 261,5 66416820 53008610 66416670 53002690 08.06.200725 0,2 89,5 66497360 53096590 66497210 53092130 10.06.200726 1,1 253 66507170 53054010 66508620 53059330 09.06.200727 0,4 177,5 66523900 53000900 66523590 53006690 08.06.200728 1,1 285,5 66526330 52518380 66521050 52519480 09.06.200729 1,2 347,5 66529820 52439680 6653060 52434060 09.06.200732 1,1 302 66492170 53176180 66494180 53168110 10.06.200733 1,2 263,5 66476920 53211610 66478370 53219880 10.06.200734 1,1 188,5 66462910 53253110 66464220 53245480 10.06.200735 2,1 266,5 66459960 53326890 66458720 53330940 11.06.200742 2,3 421 66441540 51025570 66439690 54024710 12.06.200744 0,8 77,5 66534000 54027340 66535030 54023540 11.06.200745 0,7 167,5 66529070 53568520 66529290 53575150 11.06.200746 0,7 140 66511630 53587910 66514280 53586630 11.06.200747 0,9 338 66465690 53464310 66466950 53467200 11.06.200748 2,1 206 66460290 53400760 66460890 53404680 11.06.200749 1,2 124,5 66490209 53469480 66491819 53467130 11.06.200750 0,8 214,5 66512990 53532650 66514800 53535810 11.06.200751 1,3 231,5 66536380 53445820 66538290 53444590 04.06.200752 1,5 323,5 66545300 53413820 66544910 53409140 04.06.200754 1,5 347 66541830 53332690 66542300 53338910 04.06.200755 1,9 617,5 66540940 53236740 66540250 53241270 04.06.200758 1,4 227 66540270 53137000 66539990 53142090 04.06.200760 1,8 377,5 66546010 53054920 66545780 53059200 04.06.2007

500 2,8 319,5 66219430 54502870 66221160 54499680 06.06.2007501 2,6 388,5 66319690 54242180 66321390 54238910 06.06.2007502 2,1 278,5 66311320 54358990 66311090 54364870 06.06.2007503 1,7 227 66379430 54184990 66378150 54188830 13.06.2007504 2,4 349 66357230 54224230 66355150 54227200 13.06.2007505 2,0 256,5 66290990 54427280 66288970 54431160 06.06.2007506 2,8 337 66237930 54450740 66236580 54446500 06.06.2007507 2,0 276,5 66280000 54221320 66281550 54218150 06.06.2007508 216,5 66186000 54490280 66184990 54497280 06.06.2007509 2,5 199,5 66258770 54566160 66257820 5457232 06.06.2007510 419 66434060 53071960 66431910 53072390 12.06.2007511 1,7 250 66330990 54026860 66329230 54026380 14.06.2007512 325,5 66362500 53591340 66364090 53588580 14.06.2007513 2,1 359,5 66391020 54127930 66389620 54131190 13.06.2007514 2,2 402,5 66421480 54068370 66419470 54070890 12.06.2007

Appendix 2

Alignment of all own Hematodinium sp. Sequences 0 10 20 30 40 50 60 70 80 90 100 110 #FE_51_4b CAGTTTCTGG AAGTGGCAGC TGGAAGTTTA GTGAACCTTA TCACTTAGAG GAAGGAGAAG TCGTAACAAG GTTTCCGTAG GTGAACCTGC GGAAGGATCA TTCGCACGAA TAATCAATAA #FE_14_16b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_23_HC_6a ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_23_12 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_23_13 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_24_1b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_24_3b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_24_9b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_27_5b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_43_4b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_43_16b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---..G.... .......... #FE_43_19b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---..G.... .......... #FE_44_3a ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_44_5b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_44_12b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_44_14b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_47_1b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_47_3b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_47_10b ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_47_17a ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---....... .......... #FE_52_5b .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_12a .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_3a .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_1a .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... #FE_51_6b .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... #FE_51_5b .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... #FE_51_1a .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... 120 130 140 150 160 170 180 190 200 210 220 230 #FE_51_4b AAAACACCGT GAACCTTGGC CATTAGCACG AGCAAAAAA- GCGCATGCGC ATGCTGCATG CCCCCGCCGC CGCCTCCGCT GTGTGTGTGT GGGGGTGTTT GTGTGTGCGC GTTCGTGCTA #FE_14_16b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_23_HC_6a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_23_12 .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_23_13 .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_24_1b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_24_3b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_24_9b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_27_5b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_43_4b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_43_16b ...-.G.... T......... .......G.. .A.......A .......... ....------ --........ ...T.----- -......... ----..T.G. .....C..T. ...G.C.... #FE_43_19b ...-.G.... T......... .......G.. .A.......A .......... ....------ --........ ...T.----- -......... ----..T.G. .....C..T. ...G.C.... #FE_44_3a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_44_5b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_44_12b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_44_14b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_47_1b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_47_3b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_47_10b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_47_17a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_5b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_12a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_3a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_52_1a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_51_6b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_51_5b .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... .......... #FE_51_1a .......... .......... .......... .........- .......... .......... .......... .......... .......... .......... .......... ..........

240 250 260 270 280 290 300 310 320 330 340 350 #FE_51_4b CTAAGGGCTG TGAGTGATGG GGAACCACCT CTCCAAATAT TTCT-CCAGC CCACGTTTGT TTTCCTTATA ATAACTCTCT AATTTCA-CT TATTCAATTA TATAAC-TAA GCTTCTTCTC #FE_14_16b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_23_HC_6a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_23_12 .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_23_13 .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_24_1b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......A... .......... #FE_24_3b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_24_9b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_27_5b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_43_4b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_43_16b .......... .......G.. ..G....... .......... CC.CA..... ....T..... ..C..A.... .......... ....C..-.. .......... ..--..-... ..C------. #FE_43_19b .......... .......G.. ..G....... .......... CC.CA..... ....T..... ..C..A.... .......... ....C..T.. .......... ..--..-... ..C------. #FE_44_3a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_44_5b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_44_12b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_44_14b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_47_1b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_47_3b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_47_10b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_47_17a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_52_5b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_52_12a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_52_3a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_52_1a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_51_6b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_51_5b .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... #FE_51_1a .......... .......... .......... .......... ....-..... .......... .......... .......... .......-.. .......... ......-... .......... 360 370 380 390 400 410 420 430 440 450 460 470 #FE_51_4b CCCTTCCCTT CTTCGTCCAG AAGAAGAAGG AGGAGGAGGA ------GGAG GGAGGTTATA TATATAATTT T-CAATTTAG AAAATTTTAG CGATGAATGC CTCGGCTCGG GTTA #FE_14_16b .......... .NNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N-NNNNNNN- ---------- ---------- ---------- ---- #FE_23_HC_6a .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_23_12 .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_23_13 .......... .......... .......... .......... GGAGGA.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_24_1b .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_24_3b .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_24_9b .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_27_5b .......... .......... .......... .......... ------.... ...NNNNNNN NNNNNNNNNN N-NNNNNNN- ---------- ---------- ---------- ---- #FE_43_4b .......... .......... .......... .......... GGAGGA.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_43_16b .......... .......... ......-CA. ---------- ------.... .......... .......... C-.....C.- ---------- ---------- ---------- ---- #FE_43_19b .......... .......... .......CA. ---------- ------.... .......... .......... C-.....C.- ---------- ---------- ---------- ---- #FE_44_3a .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_44_5b .......... .......... .......... .......... ------.... .......... .......... .-..N....- ---------- ---------- ---------- ---- #FE_44_12b .......... .......... .......... .......... ------.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_44_14b .......... .......... .......... .......... ------.... .......... .......... .-NNNNNNN- ---------- ---------- ---------- ---- #FE_47_1b .......... .......... .......... .......... GGAGGA.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_47_3b .......... .......... .......... .......... GGAGGA.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_47_10b .......... .......... .......... .......... GGAGGA.... .......... .......... .-.....NN- ---------- ---------- ---------- ---- #FE_47_17a .......... .......... .......... .......... GGAGGA.... .......... .......... .-.......- ---------- ---------- ---------- ---- #FE_52_5b .......... .......... .......... .......... ------.... .......... .......... .-........ .......... .......... .......... .... #FE_52_12a .......... .......... .......... .......... ------.... .......... .......... .-........ .......... .......... .......... .... #FE_52_3a .......... .......... .......... .......... ------.... .......... .......... .-........ .......... .......... .......... .... #FE_52_1a .......... .......... .......... .......... ------.... .......... .......... .-........ .......... .......... .......... .... #FE_51_6b .......... .......... .......... .......... ------.... .......... .......... .T........ .......... .......... .......... .... #FE_51_5b .......... .......... .......... .......... ------.... .......... .......... .-........ .......... .......... .......... .... #FE_51_1a .......... .......... .......... .......... ------.... .......... .......... .-........ .......... .......... .......... ....

Appendix 3

Sequence alignment of Hematodinium 0 10 20 30 40 50 60 70 80 90 100 110 120 130 #FE_52_3a_{Gp_1} GCACGAATAA T--------- ---------- -CAATAAAAA A-C----ACC -GTGAACC-T TGGCCATTAG C--------- -----A-CGA GCAAAAAA-G CGCATGCGCA TGC-TGCATG CCCCCGCCGC CGCC------ #FE_24_1b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_52_1a_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_51_1a_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_23_HC_6a_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_44_14b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_51_4b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_52_12a_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_51_5b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_24_3b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_47_10b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_44_5b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_44_12b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_47_17a_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_24_9b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_23_13_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_47_3b_{Gp_1} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #DQ871211_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031997_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031983_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....GCCGCC #EF031974_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....GCCGCC #EF031978_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF032001_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031994_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096216_P_bernhardus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031969_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #N_norvegicus_DQ871212_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #DQ084246_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....GCCGCC #EF031976_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096198_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096196_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF032013_P_bernhardus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...A...... .......... ....------ #EF032003_C_opilio_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031975_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....GCCGCC #DQ084245_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....GCCGCC #EF031977_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF032002_C_opilio_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031990_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031967_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096197_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031966_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096200_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031968_N_norvegicus_{Gp_2} ..G....... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096208_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096211_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096205_P_bernhardus_{Gp_2} .......... .--------- ---------- -......... .A.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096202_P_bernhardus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096203_P_bernhardus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096225_C_maenas_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF032004_C_opilio_{Gp_2} .......A.. .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... ........A. ....------ #EF031985_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF032008_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031989_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EF031971_N_norvegicus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096199_C_pagurus_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096220_C_maenas_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096222_C_maenas_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096223_C_maenas_{Gp_2} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #FE_43_19b_{Gp_3} ..G....... .--------- ---------- -......... --.----G.. -..T....-. .......... .--------- -----G-... A.......A. .......... ...------- --........ ...T------ #FE_43_16b_{Gp_3} ..G....... .--------- ---------- -......... --.----G.. -..T....-. .......... .--------- -----G-... A.......A. .......... ...------- --........ ...T------ #EU096217_Munida_rugosa_{Gp_4} .......... .--------- ---------- -......... .-.----... -.......A. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096218_P_prideaux_{Gp_4} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #EU096219_P_prideaux_{Gp_4} .......... .--------- ---------- -......... .-.----... -.......-. .......... .--------- -----.-... ........-. .......... ...-...... .......... ....------ #DQ925234_Uncultured_clone_{Gp_5} .......G.. AAATAATATA TTTTATTATT TTCGC.C.C. .A.ATTC... -.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. T-GGG...GT G.TG..TTG. TTA.TA.T.. TA.T-TCTTA #DQ925236_Uncultured_clone_{Gp_5} .......G.. AAATAATATA TTTTATTATT TTCGC.C.C. .A.ATTC... -.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. T-GGG...GT G.TG..TTG. TTA.TA.T.. TA.T-TCTTA #EF173451_S_serrata_{Gp_5} ---------- ---------- ---------- ---------- -T.ATTC... C.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. T-GGG...GC G---..TTG. TTA.TA.A.. TA.----TTA #EF173454_P_trituberculatus_{Gp_5} ---------- ---------- ---------- ---------- -T.ATTC... C.GTG...-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. T-GGG...GC G---..TTG. TTA.TA.A.. TA.----TTA #EF065708_perezi_clone_{Gp_5} .......... .---AATATA TTTTATTATT TTC.C.C.C. .A.ATTC... -.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. TTGGG...GT G---..TTG. TTA.TA.T.. TA.----TTA #EF065709_perezi_clone_{Gp_5} .......... .---AATATA TTTTATTATT TTC.C.C.C. .A.ATTC... -.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. TTGGG...GT G---..TTG. TTA.TA.T.. TA.----TTA #EF153726_perezi_clone_{Gp_5} .......... .---AATATA TTTTATTATT TTC.C.C.C. .A.ATTC... -.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. TTGGG...GT G---..TTG. TTA.TA.T.. TA.----TTA #EF153727_perezi_clone_{Gp_5} .......... .---AATATA TTTTATTATT TTC.C.C.C. .A.ATTC... -.......T. A.-....... .T-ACGACGA CTACT.G.T. ..T.CTG.-. TTGGG...GT G---..TTG. TTA.TA.T.. TA.----TTA #EF153728_perezi_clone_{Gp_5} .......... .---AATATA TTTTATTATT TTC.C.C.C. .A.ATTC... -.......-. .A........ .T-ACGACGA CTACT.G.T. ..T.CTG.-. TTGGG...GT G---..TTG. TTA.TA.T.. TA.----TTA

140 150 160 170 180 190 200 210 220 230 240 250 260 270 #FE_52_3a_{Gp_1} TCCGCTG--- ----TGTGTG TGTG------ --GGGGTGTT TGTGTGTGCG CGTTCGTGCT ACTAAGGGCT GTGAG----T GATGGGGAAC CACCTCTCCA AAT-ATTTCT -CCAGCCCAC GTTTGTTTTC CTTATA-ATA #FE_24_1b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_52_1a_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_51_1a_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_23_HC_6a_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_44_14b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_51_4b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_52_12a_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_51_5b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_24_3b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_47_10b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_44_5b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_44_12b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_47_17a_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_24_9b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_23_13_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #FE_47_3b_{Gp_1} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #DQ871211_C_pagurus_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031997_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031983_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031974_N_norvegicus_{Gp_2} .......TG- TGTG...... ..-------- --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031978_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF032001_C_pagurus_{Gp_2} .......TG- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031994_C_pagurus_{Gp_2} .......TG- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096216_P_bernhardus_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031969_N_norvegicus_{Gp_2} .......TG- TGTG...... ....TGTGTG TG........ .......... .......... .......... .....----. .......... .......... ...-...... -..G...... .......... ......-... #N_norvegicus_DQ871212_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #DQ084246_N_norvegicus_{Gp_2} .......TG- TGTG...... ....T----- -G........ .......... .......... .......... .....----. .......... .......... ...-...... C.AG-..... .......... ......-... #EF031976_N_norvegicus_{Gp_2} .......TG- TG--...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096198_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096196_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF032013_P_bernhardus_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF032003_C_opilio_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031975_N_norvegicus_{Gp_2} .......TG- TGTG...... ..-------- --........ ........T. .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #DQ084245_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----A .......... .......... ...-...... C.AG...... .......... ......-... #EF031977_N_norvegicus_{Gp_2} .......TG- TG--...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF032002_C_opilio_{Gp_2} .....----- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031990_C_pagurus_{Gp_2} .......TG- TG--...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031967_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096197_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031966_N_norvegicus_{Gp_2} .......TG- TG--...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096200_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031968_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --......C. .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096208_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096211_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096205_P_bernhardus_{Gp_2} .......TG- TGTG....-- ---------- --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096202_P_bernhardus_{Gp_2} .......TG- TGTG....-- ---------- --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096203_P_bernhardus_{Gp_2} .......TG- TGTG....-- ---------- --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096225_C_maenas_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF032004_C_opilio_{Gp_2} .......--- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -.......G. .......... ......-... #EF031985_C_pagurus_{Gp_2} .......TG- TG--...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF032008_N_norvegicus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031989_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EF031971_N_norvegicus_{Gp_2} .......TG- TGTG...... ....TGTGTG --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096199_C_pagurus_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096220_C_maenas_{Gp_2} .......TG- TG--...... ....------ --........ .......... .......... ..C....... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096222_C_maenas_{Gp_2} .......TG- ----...... ....------ --........ .......... .......... .......... .....----. .......... .......... ...-...... -......... .......... ......-... #EU096223_C_maenas_{Gp_2} .......TG- TGTG...... ....------ --........ .......... .......... .......... .....----. .......... .N........ ...-...... -......... .......... ......-... #FE_43_19b_{Gp_3} .--------- ----...... ...------- -----..T.G ......C..T ....G.C... .......... .....----. ..G....G.. .......... ...-..CC.C A......... T.......C. .A....-... #FE_43_16b_{Gp_3} .--------- ----...... ...------- -----..T.G ......C..T ....G.C... .......... .....----. ..G....G.. .......... ...-..CC.C A......... T.......C. .A....-... #EU096217_Munida_rugosa_{Gp_4} .......--- ----...... ....------ --.......G .......... ..C...C... ........G. .....----A .......... .C........ ...-...... -...C..... ..G....... ......-... #EU096218_P_prideaux_{Gp_4} .......--- ----...... ....------ --........ .......... .......... ........G. .....----. .......... .......... ...-...... -...C..... .......... ......-... #EU096219_P_prideaux_{Gp_4} .......--- ----...... ....------ --........ .......... .......... ........G. .....----. .......... .......... ...-...... -...C..... .......... ......-... #DQ925234_Uncultured_clone_{Gp_5} CT..TA.CT- GAAC..CACA CACACTAGTA -CCCCTCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #DQ925236_Uncultured_clone_{Gp_5} CT..TA.CT- GAAC..CACA CACACTAGTA -CCCCTCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF173451_S_serrata_{Gp_5} C...TA.CTT GAAC.--ACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF173454_P_trituberculatus_{Gp_5} C...TA.CT- GAAC.--ACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF065708_perezi_clone_{Gp_5} CT..TA.CT- GAGC.ACACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF065709_perezi_clone_{Gp_5} CT..TA.CT- GAGC.ACACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF153726_perezi_clone_{Gp_5} CT..TA.CT- GAGC.ACACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF153727_perezi_clone_{Gp_5} CT..TA.CT- GAGC.ACACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C. #EF153728_perezi_clone_{Gp_5} CT..TA.CT- GAGC.ACACA CACACTAGTA -CCT.TCTC. ..CTG..AG. A.AAGTA... T...C...G. .....GGTAC .G...TAGTA ...GC..A.C .C.G.AC..C T...T..... .....C.... .A..A.C.C

280 290 300 310 320 330 340 350 360 370 380 390 400 410 #FE_52_3a_{Gp_1} AC-TCTCTAA TTTCA-CTTA TTCAA-TTAT ATAAC-TAAG CTTCTTCTCC CCTTCCCTTC TTCGTCCAGA AGAAGAAGGA G---GAGGAG GA------GG AGGGAGGTTA TATATATAAT TTT-CAATTT AGAAAA #FE_24_1b_{Gp_1} ..-....... .....-.... .....-.... .....A.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .----- #FE_52_1a_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #FE_51_1a_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #FE_23_HC_6a_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .----- #FE_44_14b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-NNNNNN N----- #FE_51_4b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #FE_52_12a_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #FE_51_5b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #FE_24_3b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .----- #FE_47_10b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..GGAGGA.. .......... .......... ...-.....N N----- #FE_44_5b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-..N... .----- #FE_44_12b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .----- #FE_47_17a_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..GGAGGA.. .......... .......... ...-...... .----- #FE_24_9b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .----- #FE_23_13_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..GGAGGA.. .......... .......... ...-...... .----- #FE_47_3b_{Gp_1} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..GGAGGA.. .......... .......... ...-...... .----- #DQ871211_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #EF031997_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........-- ----...... ..GGAGGA.. ---....... .......... ...-...... ...... #EF031983_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. .......... .......... ...-...... ...... #EF031974_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. .......... .......... ...-...... ...... #EF031978_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. ---....... .......... ...-...... ...... #EF032001_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .....- #EF031994_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... ..T....... .......... .---...... ..------.. .......... .......... ...-...... .....- #EU096216_P_bernhardus_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #EF031969_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. ---....... .......... ...-...... ...... #N_norvegicus_DQ871212_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #DQ084246_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .------.G. ..GGAGGA.. ...A------ ---------- ---------- ------ #EF031976_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .AAG...... ..GGAGGA.. .......... .......... ...-...... ...... #EU096198_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .A..A...-- ----...... ..GGAGGA.. ---....... .......... ...-...... ...... #EU096196_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .A..A...-- ----...... ..GGAGGA.. ---....... .......... ...-...... ...... #EF032013_P_bernhardus_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... ...... #EF032003_C_opilio_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .C........ .......... .---...... ..------.. .......... .......... ...-...... ...... #EF031975_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. .......... .......... ...-...... ...... #DQ084245_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .------... ..GGAGGA.. ...A------ ---------- ---------- ------ #EF031977_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .AAG...... ..GGAGGA.. .......... .......... ...-...... ...... #EF032002_C_opilio_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. .......... .......... ...-...... ...... #EF031990_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .....- #EF031967_N_norvegicus_{Gp_2} ..-....... ..C..-.... .....-.... .--..-.... .......... .......... .......... ........A. .AAG...... ..GGAGGA.. .......... .......... ...-...... ...... #EU096197_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... ---------. ..GGAGGA.. .......... .......... ...-...... ...... #EF031966_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .---...... ..GGAGGA.. .......... .......... ...-...... ...... #EU096200_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... ---------. ..GGAGGA.. .......... .......... ...-...... ...... #EF031968_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .AAG...... ..GGAGGA.. .......... .......... ...-...... ...... #EU096208_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. .AAG...... ..GGAGGA.. .......... .......... ...-...... ...... #EU096211_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. ---G...... ..GGAGGA.. .......... .......... ...-.....- ...... #EU096205_P_bernhardus_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... ---------. ..GGAGGA.. .......... .......... ...-.....- ...... #EU096202_P_bernhardus_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... ---------. ..GGAGGA.. .......... .......... ...-.....- ...... #EU096203_P_bernhardus_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .......... ---------. ..GGAGGA.. .......... ......C... ...-.....- ...... #EU096225_C_maenas_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... ........G. ..C-...... .....- #EF032004_C_opilio_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .------... ..------.. .......... .......... ..C-...... .....- #EF031985_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-T..... .....- #EF032008_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. ---G...... ..GGAGGA.. .......... .......... ...-...... ...... #EF031989_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .....- #EF031971_N_norvegicus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... ........A. ---G...... ..GGAGGA.. .......A.. .......... ...-...... ...... #EU096199_C_pagurus_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... ---------. ..GGAGGA.. .......... .......... ...-...... ...... #EU096220_C_maenas_{Gp_2} ..-....... .....-.... .....-.... .....-.... .......... .......... .......... .A........ .---...... ..------.. .......... .......... ...-...... .....- #EU096222_C_maenas_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .....C.... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .....- #EU096223_C_maenas_{Gp_2} ..-....... .....-.... .....-.... .--..-.... .......... .......... .......... .......... .---...... ..------.. .......... .......... ...-...... .....- #FE_43_19b_{Gp_3} ..-....... ..C..T.... .....-.... .--..-.... .C------.. .......... .......... ......CA.- ---------- --------.. .......... .......... ..C-.....C .----- #FE_43_16b_{Gp_3} ..-....... ..C..-.... .....-.... .--..-.... .C------.. .......... .......... .....-CA.- ---------- --------.. .......... .......... ..C-.....C .----- #EU096217_Munida_rugosa_{Gp_4} ..-....... .....-.... .....-.... .....-A... .......... .....T..A. AAAA....C. .A..A..A.. .---...... ..------.. .......... .......... ...-...... .....- #EU096218_P_prideaux_{Gp_4} ..-....... .....-.... .....-.... .....-.... .......... ........A. AAAA....C. .A..A..A.. .---...... ..------.. .......... .......... ...-...... .....- #EU096219_P_prideaux_{Gp_4} ..-....... .....-.... .....-.... .....-.... .......... ........A. AAAA....C. .A..A..A.. .---...... ..------.. .......... .......... ...-...... .....- #DQ925234_Uncultured_clone_{Gp_5} ..A....... .....-GC.. ....T----- ----.----- ---------- -T.G.T..G. .C.C.TTC.C G.GGAT...G C--------- ---------- ------T..C .TC.A.CG-. A.G----AC. .....- #DQ925236_Uncultured_clone_{Gp_5} ..A....... .....-GC.. ....T----- ----.----- ---------- -T.G.T..G. .C.C.TTC.T G.GGAT...G C--------- ---------- ------T..C .TC.A.CG-. A.G----AC. .....- #EF173451_S_serrata_{Gp_5} ..A....... .....-A... ....T----- ----.----- ---------- -T.G.T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..C .TC.A.CGG. A.G----AC. ...--- #EF173454_P_trituberculatus_{Gp_5} ..A....... .....-A... ....T----- ----.----- ---------- -T.G.T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..C .TC.A.CGG. A.G----AC. ...--- #EF065708_perezi_clone_{Gp_5} ..A....... .....-A... ....T----- ----.----- ---------- -T.G.T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..T .TC.A.CG-. A.G----AC. .....- #EF065709_perezi_clone_{Gp_5} ..A....... .....-A... ....T----- ----.----- ---------- -T.G.T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..T .TC.A.CG-. A.G----AC. .....- #EF153726_perezi_clone_{Gp_5} ..A....... .....-A... ....T----- ----.----- ---------- -T.G.T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..T .TC.A.CG-. A.G----AC. .....- #EF153727_perezi_clone_{Gp_5} ..A....... .....-A... ....TC..G- --------C- ---------- -----T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..T .TC.A.CG-. A.G----AC. .....- #EF153728_perezi_clone_{Gp_5} ..A....... .....-A... ....T----- ----.----- ---------- -T.G.T..G. .C.C.TT..C G.GGTT...G C--------- ---------- ------T..T .TC.A.CG-. A.G----AC. .....-

Appendix 4

18S Alignment: Hematodinium sp., Hematodinium perezi, Outliner sequence 0 10 20 30 40 50 60 70 80 90 100 #FE_52_5b CAGTTTCTGG AAGTGGCAGC TGGAAGTTTA GTGAACCTTA TCACTTAGAG GAAGGAGAAG TCGTAACAAG GTTTCCGTAG GTGAACCTGC GGAAGGATCA TTCG #EF153727_perezi_clone .......... ...C...... .......... .......... .......... .......... .......... .......... .......... .......... .... #FE_43_16b .......... .......... ......G... .C........ C......... .....T.... .......... .C........ ...G.A.... .......... ....

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