Journal of Fish Diseases 2005, 28, 331–338
Polymorphisms in the sequences of Marteilia internal
transcribed spacer region of the ribosomal RNA genes
(ITS-1) in Spain: genetic types are not related with bivalve
hosts
B Novoa1, D Posada2 and A Figueras1
1 Instituto de Investigaciones Marinas, CSIC, Vigo, Spain
2 Facultad de Ciencias, Universidad de Vigo, Campus Universitario Lagoas-Marcosende, Vigo, Spain
Abstract
Marteilia refringens is a protozoan parasite causing a
disease notifiable to the Office International des
Epizooties (OIE) and its distribution has implications for the transfer of live animals. The internal
transcribed spacer-1 (ITS-1) from Marteilia clones
contains polymorphism. Digestion with HhaI
reveals two different restriction profiles, previously
referred as ÔOÕ (Marteilia from oyster or Marteilia
refringens) and ÔMÕ (Marteilia from mussels or
Marteilia maurini). The aim of the present work
was to determine whether the two previously described Marteilia molecular types (O and M) exist in
the Iberian Peninsula and the strictness of the
association with their bivalve host species. The
sequence variability in the ITS-1 of Marteilia species
was studied in mussels, Mytilus galloprovincialis, and
flat oysters, Ostrea edulis, from different geographical locations in Spain, to establish the existence and
the distribution of different species or molecular
types. Although there were two distinct evolutionary lineages that corresponded more or less strictly
with the ÔMÕ and ÔOÕ types, it was evident from the
estimated phylogeny that some ÔOÕ types have
switched to ÔMÕ type, and vice versa. Moreover, ÔOÕ
types were found in mussels and ÔMÕ types were
found in oysters, which suggests that there have
been several cross-species transmissions of Marteilia
between mussels and oysters.
Correspondence A Figueras, Instituto de Investigaciones
Marinas, CSIC, Eduardo Cabello 6, 36208 Vigo, Spain
(e-mail: [email protected])
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Keywords: bivalve, ITS, Marteilia maurini, Marteilia refringens, Mytilus galloprovincialis, Ostrea edulis.
Introduction
Marteilia refringens is a member of the Phylum
Paramyxea (Berthe, Le Roux, Peyretaillade, Peyret,
Rodriguez, Gouy & Vivares 2000), associated with
serious and recurrent mortalities in the European
flat oyster, Ostrea edulis. The disease caused by this
parasite is currently listed by the Office International des Epizooties (OIE) (2000) as a notifiable
disease.
Although the production of flat oysters in the
European aquaculture is relatively low (5000 ton
year)1), infection by M. refringens still has serious
consequences. The presence of M. refringens in
other bivalve species such as mussels, Mytilus
galloprovincialis (production 650 000 ton year)1),
means that these species are effectively considered as
carriers for the pathogen, and as a consequence their
movement between countries can be restricted.
Marteilia refringens has been found in France,
Greece, Italy, Morocco, Portugal and Spain. In its
early developmental stages, the parasite is 5–8 lm
in size reaching 40 lm during sporulation. In the
bivalve host, the life cycle takes place mainly in the
digestive tubules and in the epithelia of stomach
and intestine. Interestingly, whilst the prevalence of
Marteilia has remained stable in France, it has
almost disappeared in Spain.
Two different species of Marteilia have been
reported in Europe, M. refringens, parasitic in the
B Novoa et al. Marteilia polymorphisms in Spain
Journal of Fish Diseases 2005, 28, 331–338
flat oyster, O. edulis (Grizel, Comps, Bonami,
Cousserans, Duthoit & Le Pennec 1974), but also
described in mussels, M. galloprovincialis (Villalba,
Mourelle, Carballal & Lopez 1993), and
M. maurini, found in M. galloprovincialis (Comps,
Pichot & Papagianni 1982). In both cases light and
transmission electron microscopy were used to
establish the parasite species. The differences
between both species have traditionally been based
on ultrastructural characteristics and host specificity. However, a recent study has shown that these
ultrastructural characteristics are not sufficient to
distinguish M. refringens and M. maurini (Longshaw, Feist, Matthews & Figueras 2001).
At a molecular level, although the sequence of the
18S rRNA gene does not allow discrimination
between Marteilia isolates (Berthe et al. 2000), it is
possible to find genetic polymorphisms in the
internal transcribed spacer (ITS) region, associated
with the host shellfish species (Le Roux, Lorenzo,
Peyret, Audemard, Figueras, Vivares, Gouy &
Berthe 2001). Based on RFLP of the ITS-1 PCR
products, Le Roux et al. (2001) showed the existence of two profiles, ÔOÕ and ÔMÕ, found almost
exclusively in oysters and mussels, respectively (only
in one case, a flat oyster, where both types were
found in the same animal). Hence, these authors
suggested the occurrence of two Marteilia species in
Europe: M. maurini, a non-notifiable pathogen that
infects mussels, and M. refringens, a notifiable
pathogen that infects oysters. Therefore, the transfer
of live mussels would not be affected by international regulations concerning notifiable diseases.
The objective of this work was to establish the
occurrence and distribution of different species or
molecular types of Marteilia in different hosts and
geographical locations in Spain, a country with a
high bivalve production and little studied by Le
Roux et al. (2001). To accomplish this goal, the
variability of the ITS-1 sequence from Marteilia
strains infecting oysters and mussels was examined.
Ría de Vigo
Ría de Arosa
(N-W)
PORTUGAL
SPAIN
Delta del Ebro
(N-E)
Huelva
(S-W)
Figure 1 Map of Spain showing the locations from which
bivalves were sampled.
As a very low prevalence of Marteilia was
observed during the sampling period, infected
animals were also obtained from paraffin blocks.
Formalin-fixed and paraffin-embedded infected
O. edulis were obtained from Delta del Ebro
(Mediterranean Coast).
DNA extraction
The prevalence of Marteilia was checked using a
Giemsa modified staining method (Hemacolor Kit;
Merck, Whitehouse Station, NJ, USA) on bivalve
digestive gland imprints. More than 2000 animals
were screened to select infected individuals.
DNA was extracted from Marteilia sp.-infected
animals. Digestive gland fragments were added to
500 lL of extraction buffer (NaCl 100 mm, EDTA
25 mm, pH 8, SDS 0.5%) with proteinase K
(100 mg mL)1). After an overnight incubation at
50 C, DNA was extracted using a standard
protocol involving phenol/chloroform, precipitated
with ethanol, and treated with RNase A.
When infected animals were obtained fixed and
embedded in paraffin blocks, sections were cut at
10–20 lm and transferred to microfuge tubes.
Approximately 5–10 tissue sections were deparaffinized by successive washes in xylene and ethanol
and used for DNA extraction.
Materials and methods
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Animals
Amplification, sequencing and RFLP typing
Infected bivalves (O. edulis, M. galloprovincialis)
were obtained from well-known endemic zones of
marteiliosis in Spain (Fig. 1). Mytilus galloprovincialis were collected from Rı́a de Arosa and Rı́a de
Vigo (North West coast). Ostrea edulis were
collected from Huelva (South West coast).
The ITS-1 region was amplified via the polymerase
chain reaction (PCR) using specific primers previously described (Le Roux et al. 2001). PCR
reactions were performed in 25 lL using standard
conditions with a final concentration of 3 mm
MgCl2 in a Applied Biosystems 2400 cycler
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B Novoa et al. Marteilia polymorphisms in Spain
Journal of Fish Diseases 2005, 28, 331–338
(Applied Biosystems, Foster City, CA, USA).
Reactions not containing DNA were also carried
out as negative controls. Following an initial
denaturation at 94 C reactions were subjected to
30 cycles of denaturation at 94 C for 1 min,
annealing at 55 C for 1 min and elongation at
72 C for 1 min. The final extension lasted 10 min
at 72 C.
PCR products were cloned directly in the vector
pCR 2.1 following the standard protocol supplied by
the manufacturer (TA Cloning Kit; Invitrogen,
Carlsbad, CA, USA). Amplified fragments were
ligated into linearized pCR 2.1 vector overnight at
14 C, using DNA ligase and used to transform
competent Escherichia coli TOP10 F¢ cells. Screening
of clones carrying the ITS fragment was performed
by PCR using ITS-specific primers and adding
positive colonies to the PCR mixture reaction. To
confirm the results, positive clones were rescreened
by agarose gel electrophoresis of recombinant DNA,
obtained from alkaline lysis minipreps with and
without digestion with endonuclease EcoRI.
Polymorphism in the ITS region amplified by
PCR was investigated by RFLP analysis. Amplified
products were digested with restriction endonuclease HhaI and separated on a 2% agarose gel.
PCR products were purified by digestion with
exonuclease and shrimp phosphatase (SAP) for 1 h
at 37 C and 15 min at 80 C to inactivate the
enzymes. Direct sequencing of purified PCR products was performed using a BigDye Terminator
Cycle Sequencing Ready Reaction Kit (Applied
Biosystems), according to the manufacturer’s directions, in an ABI PRISM 377 automated sequencer
(Applied Biosystems). PCR products were
sequenced at least twice.
Phylogenetic analysis
ITS-1 sequences were aligned with the program
Clustal W (Thompson, Higgins & Gibson 1994)
and adjusted by eye. The resulting alignment was
355 nucleotides long and did not include any gaps.
The best-fit model of nucleotide substitution was
selected using the Akaike information criterion
(AIC) (Akaike 1974), as implemented in the
program Modeltest 3.2 (Posada & Crandall
1998). A phylogeny was estimated using Bayesian
methods. The Bayesian framework allows the
incorporation of phylogenetic uncertainty in the
analysis. Inferences are averaged over all possible
trees, and the result is weighted by the posterior
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probability that the tree is correct. Trees are
sampled according to their posterior probability
using Markov Chain Monte Carlo (MCMC)
techniques (Huelsenbeck, Rannala & Masly 2000;
Huelsenbeck, Ronquist, Nielsen & Bollback 2001;
Huelsenbeck, Larget, Miller & Ronquist 2002).
This analysis was carried out using the program
Mr.Bayes 3.0b4 (Huelsenbeck & Ronquist 2001;
Huelsenbeck et al. 2002). Four MCMC chains
were run, three heated and one cold for 1 · 107
generations, with trees being sampled every 100
generations. Convergence was obtained very quickly
and after the run, the first 200 trees (20 000
generations) were discarded as ÔburninÕ. A 50%
majority rule consensus tree was computed from all
99 800 trees sampled. The posterior probabilities
calculated were used to assess the credibility of a
series of hypotheses on the distribution and origins
of the Marteilia samples.
Results
PCR typing of Marteilia species and strains
The ITS region from the different Marteilia clones
included several polymorphisms (Fig. 2). In addition, restriction analysis after HhaI digestion
showed two different profiles, previously referred
as ÔOÕ and ÔMÕ (Le Roux et al. 2001) (Fig. 3).
In N.W. Spain, 38 Marteilia clones were isolated
from 13 mussels. Of these, 79% were type ÔMÕ and
21% were type ÔOÕ. In Delta del Ebro, 16 Marteilia
clones were isolated from five oysters. Of these,
12% were type ÔMÕ and 88% were type ÔOÕ. Two
oysters were coinfected by both ÔOÕ and ÔMÕ types.
In Huelva, 26 Marteilia clones were isolated from
seven oysters. Of these, 61% were type ÔMÕ and
29% were type ÔOÕ. No coinfection was detected
except in the case of a new RFLP type (termed ÔXÕ)
isolated from an oyster infected with the ÔMÕ type.
Examination of the ITS sequences (GenBank
accession numbers AY324551–AY324588) permitted the identification of groups of Marteilia strains
(Fig. 3). Indeed, these two groups coincided with
two distinct and independent evolutionary lineages
(Fig. 4).
Group or type 1 (previously called ÔMussel typeÕ
by Le Roux et al. 2001) consists of Marteilia strains
mainly from mussels, but also from oysters. Group
or type 2 (previously called ÔOyster typeÕ by Le
Roux et al. 2001) consists of Marteilia strains
mainly found in oysters, but also in mussels. In
Journal of Fish Diseases 2005, 28, 331–338
B Novoa et al. Marteilia polymorphisms in Spain
Figure 2 Small fragment of Marteilia ITS sequences showing differences between the two types (shaded) and restriction sites for HhaI
(boxed).
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B Novoa et al. Marteilia polymorphisms in Spain
Journal of Fish Diseases 2005, 28, 331–338
(a)
M
1
2
3
4
5
6
7
8
(b)
1
2
3
4
5
6
7
8
Figure 3 Agarose gels showing different profiles obtained after
digestion of the amplified ITS fragment with the restriction
enzyme HhaI. Gel a: M, marker; lanes 1–4: different clones of
Marteilia ITS from Mytilus galloprovincialis Rı́a de Vigo with ÔM
profileÕ; lanes 5–8: different clones of Marteilia ITS from Ostrea
edulis collected at Delta del Ebro showing an ÔMÕ profile (lane 5)
and ÔOÕ profile (lanes 6–8). Gel b: different clones of Marteilia
ITS from O. edulis collected at Huelva; lanes 1, 3, 4, 5, 6 with
ÔMÕ profile; lanes 7 and 8 with ÔOÕ profile and lane 2 with a
different restriction pattern corresponding to the clone Oy2-X.
general, these two groups were correlated with the
restriction profiles ÔOÕ and ÔMÕ, although two
exceptions were found. Strain Oy2X displayed a
new RFLP profile different from the ÔOÕ and ÔMÕ
profiles. Strain Oy11.10M has another restriction
site very close to the site 344 that results in an ÔMÕ
pattern, although this strain is clearly of type 2.
Phylogenetic analysis
The AIC selected the Tamura-Nei model (Tamura
& Nei 1993) with rate heterogeneity among sites
(TrN + G) as the best-fit substitution model for
this sample. Bayesian analysis indicated that many
trees are compatible with the data (the 95% credible
set contained 94 810 trees). A 50% majority rule
consensus of all sampled trees indicated the existence of two distinct lineages that correlate well,
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although not perfectly, with types ÔOÕ and ÔMÕ
(Fig. 4). To understand the implications of this
phylogeny we assessed the probability that their
strains do cluster together in the tree according to
host, RFLP type, sampling location or individual
(Table 1).
1. The posterior probability that these strains cluster
together by RFLP type (ÔOÕ or ÔMÕ) is zero, which
suggests that this RFLP type evolved more than
once. However, this result was due mainly to
strains Oy 11.10 M and Oy 2 X. If these
sequences are excluded, the posterior probability
that these strains cluster together by RFLP type is
close to 1.
2. The posterior probability that these strains cluster
together by host (oyster or mussel) is zero, which
suggests that there have been several cross-species
transmissions of Marteilia between mussels and
oysters, although without an outgroup species it
is not possible to tell in which direction.
3. The posterior probability that these strains cluster
together by location is also zero, which suggests
that there have been several migration events
across Spain.
4. The posterior probability that sequences cluster
together by clone is always very small, close to
zero, suggesting that those individuals for which
there is more than one clone were infected several
times and by different Marteilia lineages.
Discussion
The aim of this work was to evaluate the existence
of different types of Marteilia in Spain as a follow
up to previous work conducted with Marteilia from
several locations in France and one location each in
Spain and Croatia (Le Roux et al. 2001). The
occurrence of two species of this parasite in Europe,
one infecting oysters (M. refringens) and another
infecting mussels (M. maurini), was postulated by
Comps et al. (1982), when M. maurini was
described in M. galloprovincialis and M. edulis
(Auffret & Poder 1985). However, Figueras &
Robledo (1993) determined that healthy oysters
cultured together with Marteilia-infected mussels
never developed the disease. Longshaw et al. (2001)
reported that ultrastructural study of Marteilia from
different bivalves was not enough to distinguish
M. maurini from M. refringens. However, molecular
studies allowed discrimination between Marteilia
isolates from mussels and flat oysters (Le Roux et al.
2001).
B Novoa et al. Marteilia polymorphisms in Spain
Journal of Fish Diseases 2005, 28, 331–338
Mu 14.8 M
Mu 2.3 M
54
Mu 3.4 M
Mu 1.13 M
Mu 1.3 M
86
Mu 1.6 M
Mu 7.20 M
Mu 2.12 M
Mu 9.1 M
Oy 11.3 M
51
Mu 1.10 M
100
Oy 2 X
Oy 35.9 M
Oy 35.10 M
Oy 35.7 M
Mu 1.12 M
100
Mu 1.11 M
Mu 2.8 M
Mu 3.10 M
Oy 1.4 M
Mu 14.9 M
Mu 1.15 M
Mu 5.10 M
Mu 7.1 O
Oy 6.8 O
95
100
Oy 11.10 M
Oy 6.4 O
Oy 11.7 O
100
Oy 6.5 O
Oy 3.6 O
70
Oy 5.5 O
Oy 3.1 O
Oy 5.1 O
Oy 5.4 O
Mu 7.9 O
96
Mu 7.11 O
92
Oy 5.2 O
Oy 5.3 O
0.05 substitutions/site
The distribution of the ÔMÕ and ÔOÕ Marteilia
types in bivalves from N.W. Spain and Delta del
Ebro is similar to that found in another European
areas. However, this was not the case in Huelva.
Here, 61% of the Marteilia clones isolated from
oysters were ÔMÕ type. These results highlight the
idea that the correlation of ÔMÕ and ÔOÕ types to
mussels and oysters is neither perfect nor homogeneous. Indeed, this finding has important implications for the regulation of movement of mussels
across Europe as it may not be safe to transfer
Marteilia-infected mussels because they may carry
M. refringens.
Interestingly, a third ITS RFLP profile, different
from the ÔMÕ and ÔOÕ types, was found in an animal
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Figure 4 Bayesian 50% majority rule consensus phylogeny of Marteilia. Each sequence name indicates host (Mu: mussel,
Oy: oyster), individual and clone number
(7.20 is clone 20 from individual 7), RFLP
type (M or O), and sampling location ( :
Huelva, d: Rı́a de Arosa, : Rı́a de Vigo, :
Delta del Ebro). Numbers above branches
are posterior probabilities (only those above
50% are shown). Branch length scale is also
indicated.
harbouring both ÔOÕ and ÔMÕ types. Two cases of
coinfection (ÔMÕ and ÔOÕ simultaneously appearing
in the same individual) were detected in oysters and
one in mussel.
From all available information, it can be assumed
that, based on ITS-1 sequences, two main types of
Marteilia can be found. Whether this dimorphism
is related with virulence requires further study.
However, it is difficult to set up an experimental
design to assess this as all attempts to infect oysters
experimentally have been unsuccessful. The only
way to reproduce the disease is to place healthy
oysters in ponds containing infected oysters, suggesting that organisms living in the pond could act
as intermediate hosts of the parasite (Berthe, Pernas,
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Journal of Fish Diseases 2005, 28, 331–338
Table 1 Posterior probability of different phylogenetic hypotheses concerning Marteilia. The first column describes the
hypotheses assessed, which state that all sequences cluster
together (i.e. are monophyletic) according to a given criterion.
Type ÔO*Õ includes all O sequences plus Oy 11.10 M, while type
ÔM*Õ includes all M sequences except Oy 11.10 M plus Oy 2 X
Clustering
criterion
Type
ÔOÕ
ÔMÕ
ÔO*Õ
ÔM*Õ
Host
Oyster
Mussel
Location
Huelva
Delta del Ebro
Rı́a de Vigo
Clon
Mu1
Mu2
Mu3
Mu7
Mu14
Oy3
Oy5
Oy6
Oy11
Oy35
Number
of taxa
Posterior
probability
14
23
15
23
0.0000
0.0000
0.9981
0.9981
18
20
0.0000
0.0000
12
6
19
0.0000
0.0000
0.0000
7
3
2
4
2
2
5
3
3
3
0.0000
0.0009
0.0425
0.0000
0.0188
0.0672
0.0000
0.0022
0.0000
0.0000
Zerabib, Haffner, Thebault & Figueras 1998).
Recently, the copepod Paracartia (Acartia) grani
has been suggested as a possible intermediate host,
as Marteilia was detected in these organisms by
molecular techniques and also as the copepod could
be experimentally infected from infected flat oyster
(Audemard, Le Roux, Barnaud, Collins, Sautour,
Sauriau, Cousteau, Combes & Berthe 2002).
The results of the present study indicate that the
two Marteilia genetic groups are not strongly
related to the host species, in contrast to the
conclusions of the previous study of Le Roux et al.
(2001). Further studies are clearly needed on this
subject as European and OIE legislation on fish and
shellfish diseases must be built on scientific evidence, for example to determine the vector or
carrier role of mussels. Molecular techniques can
obviously help in this respect and extensive molecular studies on bivalves from different European
countries are required.
Acknowledgements
This research was supported by the research projects
FAIR CT97-3640 funded by the European Union
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and PGIDT 01MAR 40203PR funded by the
Xunta de Galicia, Spain. We thank Begoña Villaverde, Dorita Pose, Marı́a Pazos and Eva Amorı́n for
technical assistance. We also thank J. I. Navas
(CICEM, Huelva) and Dolores Furones, Institut de
Recerca i Tecnologia Agroalimentaries, San Carlos
de la Rápita Tarragona, Spain for supplying
infected oysters from the South Atlantic Coast.
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Received: 26 May 2004
Revision received: 31 March 2005
Accepted: 31 March 2005