Journal of Invertebrate Pathology 131 (2015) 43–57

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Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Marteilia spp. parasites in bivalves: A revision of recent studies

Noèlia Carrasco a,⇑, Timothy Green b, Naoki Itoh c
a
IRTA-Sant Carles de la Ràpita, Ctra. Poblenou Km 5, 43540 Sant Carles de la Ràpita, Tarragona, Spain
b
Department of Biological Sciences, Macquarie University, New South Wales 2109, Australia
c
Laboratory of Fish Diseases, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Marteilia spp. parasites are Paramyxean organisms that affect several commercial species of molluscs, and
Received 17 June 2015 thus have a socio-economical impact. These parasites also have an ecological impact on the biodiversity
Revised 29 July 2015 and population dynamics of natural mollusc beds. It has been over forty years since the first description
Accepted 30 July 2015
of Marteilia refringens, the first Marteilia species described. Despite four decades of research, the biology,
Available online 1 August 2015
cellular development, as well as the life cycle of Marteilia parasites are not fully understood. In recent
years, new studies have reported advances in knowledge of the life cycle, the description of new species,
Keywords:
the development of new molecular detection/discrimination tools, new data on biotype geographical
Marteilia sp.
M. refringens
distribution, as well as new information on host response and defence mechanisms. Such information
M. sydneyi is summarized, reported and discussed in the present review in order to facilitate a new overview of
M. cochillia the subject. However, numerous knowledge gaps are still unresolved and need to be prioritized in the
M. granula research and funding institution agendas.
M. chungmuensis Ó 2015 Elsevier Inc. All rights reserved.

1. General introduction
Marteilia spp. molluscan parasites are protozoans belonging to
the phylum, Cercozoa (Cavalier-Smith, 1998; Cavalier-Smith and
Chao, 2003), order Paramyxida (Chatton et al., 1911) and character-
ized by their particular cell-inside-cell development. The first
description of a Marteilia sp. parasite corresponded to Marteilia
refringens (Grizel et al., 1974) and was associated with mortalities
in the European flat oyster Ostrea edulis cultivated in Brittany,
France, at the end of the 1960s and early 1970s (Comps, 1970;
Herrbach, 1971). Those mortality episodes had a significant impact
on French shellfish aquaculture in terms of socio-economics,
resulting in an important decline of the flat oyster aquaculture in
Europe, which has never recovered. During a 10-year period
between the end of the 1960s and beginning of the 1980s, the
production of flat oysters in France decreased from 20,000 metric
tonnes per year to 2000, in Alderman (1979). The mortality
episodes were attributed to the action of two different protozoan
parasites: M. refringens and Bonamia ostreae (Berthe et al., 2004).
Because of the socio-economical impact of those diseases, the two
parasites, as well as Marteilia sydneyi, which causes QX disease in
the Sydney rock oyster, Saccostrea glomerata, in Australia (Perkins
⇑ Corresponding author at: IRTA, Sant Carles de la Ràpita, Ctra. Poblenou Km 5,
AP. 200, 43540 Tarragona, Spain.
E-mail address: noelia.carrasco@irta.es (N. Carrasco).
and Wolf, 1976), were thus included in the list of the OIE (World
Organisation for Animal Health) notifiable parasites, and carefully
legislated in the different affected geographical areas (Europe and
Australia) (Anonymous, 2014) in order to be controlled, managed
and their transfer prevented.
Since first detected, M. refringens and M. sydneyi have been stud-
ied using different scientific approaches and new knowledge has
been provided on their development, environmental dynamics
and life cycles. However, their complete life cycles are still
unknown, becoming one of the more ambitious milestones in this
research field. Recently, studies on the M. refringens life cycle have
pointed out a number of zooplanktonic species, including Acartia
grani and A. latisetosa, which could potentially act as an alternative
host in the life cycle of M. refringens (Audemard et al., 2002;
Carrasco et al., 2007; Boyer et al., 2013; Arzul et al., 2014).
Similarly, the polychaete Nephtys australiensis is being studied as
a potential alternative host in the life cycle of M. sydneyi (Adlard
and Nolan, 2015). Even with recent advances, many knowledge
gaps remain regarding the complex life cycles and transmission
of these parasites. Paramyxean phylogeny, biotype host range
and geographical distribution are also important points to be clar-
ified in order to better understand the impact and associated risks
related with Marteilia spp. parasites.
The use of molecular tools allowing easy and quick detection as
well as parasite molecular characterization has facilitated, in recent

http://dx.doi.org/10.1016/j.jip.2015.07.016
0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
44 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57

years, several important developments: (1) the detection of the
Marteilia spp. in new geographical areas, such as China (Wang
et al., 2012); (2) parasite characterization in new hosts such as
the razor clam Solen marginatus, the Venus clam Chamalea gallina,
the black-pigmy mussel Xenostrobus secures and the common
European cockle Cerastoderma edule (López-Flores et al., 2008a,
2008b; Pasqual et al., 2010; Carrasco et al., 2012, 2013a); and (3)
the description of new Marteilia spp. such as M. cochillia (Carrasco
et al., 2013a) in common cockle in Spain and M. granula in the
Manila clam Ruditapes philippinarum in Japan (Itoh et al., 2014),
among other findings. Thus, there are many new developments
since the last review on Marteilia spp. molluscan parasites (Berthe
et al., 2004). The aim of the present review is to summarize
the new knowledge obtained in the last 10 years, and to
highlight the priorities for future research. Furthermore, we
point out that The Paramyxean Working Group Experts Network
(www.paramyxeanworkingroup.org), has been also recently
created, and it will focus on the advance in this field of research.
2. Marteilia spp. parasite development and host response in
bivalves
2.1. Tissue affinity and host response
Marteilia spp. molluscan parasites mainly affect the digestive
gland, as in the case of M. refringens, M. sydneyi, M. cochillia, and
M. granula, or the gonadal tissue, as in the case of M. chungmuensis,
of infected bivalves and other marine invertebrates (Grizel et al.,
1974; Perkins and Wolf, 1976; Audemard et al., 2002; Carrasco
et al., 2013a; Itoh et al., 2014; Adlard and Nolan, 2015) (Fig. 1).
However, early developmental stages of Marteilia spp. parasites
have been also described in other tissues. Extrasporogonic early
stages of M. sydneyi occur in gills (Kleeman et al., 2002a; Green
et al., 2011). Early stages of M. chungmuensis are observed in the
mantle, gills and labial palps, while multiplication occurs in con-
nective tissues (Itoh et al., 2004a), and sporulation and mature
stages in the oocytes (Itoh et al., 2002).

Fig. 1. Histopathological sections of Marteilia spp. parasites in host tissues, hematoxylin and eosin staining. (A) Marteilia refringens in the digestive tubule epithelia of the
mussel, Mytilus galloprovincialis. Arrows indicate different M. refringens stages of development. Scale bar = 50 lm. (B) Marteilia cochillia in the digestive tubule epithelia of the
cockle, Cerastoderma edule. Arrows indicate different M. cochillia stages of development. Scale bar = 100 lm. (C) Marteilia sydneyi early developmental stages (arrows) in the
gill of the oyster Sacccostrea glomerata. Scale bar = 20 lm. (D). M. sydneyi mature stages (arrows) at the epithelia of the digestive tubules of S. glomerata. Scale bar = 40 lm. (E)
The ovary of the Pacific oyster, Crassostrea gigas, infected with ovarian Marteilia chungmuensis. Arrows indicate oocytes infected with the late sporulation stage. Scale
bar = 30 lm. (F) The epithelia of digestive diverticula of the Manila clam, Ruditapes philippinarum, infected with Marteilia granula. Arrows indicate sporangia containing
mature spores with eosinophilic granules. Scale bar = 10 lm.
 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
Primary stages of M. refringens usually occur in the stomach
epithelia, and have also been observed by in situ hybridization in
the epithelium of the gill lamellae of M. galloprovincialis in the
Mediterranean coast of Spain (Carrasco et al., 2008b) and in the
connective tissues of mantle, labial palps and gills in M. edulis in
Normandy, France (Garcia et al., 2009). Those locations seem to
be unusual for M. refringens, which is commonly observed, as men-
tioned before, in the digestive gland. Another atypical location was
reported for a Marteilia sp. parasite infecting the giant clam
Tridacna maxima. It was observed in the kidney tissue of its host,
inducing the formation of cyst-like foci (Norton et al., 1993).
The host tissue and cellular response to Marteilia spp. infection
can also be variable. For instance, no host reaction was observed in
O. edulis heavily infected with mature stages of M. refringens in the
digestive gland (Grizel et al., 1974; Alderman, 1979; Grizel, 1985).
Similar observations are reported for French M. edulis mussels, and
in some cases for M. galloprovincialis and O. edulis. In other
instances, hemocyte infiltration in primary and secondary diges-
tive tubules is observed in these hosts (Alderman, 1979; Robledo
and Figueras, 1995; Berthe et al., 2004), and granulocytomas and
tissue necrosis have been described in acute responses by M. gallo-
provincialis infected with M. refringens (Villalba et al., 1993a).
Interestingly, in most of the cases, M. refringens infection is associ-
ated with severe hemocyte infiltrations surrounding the parasite
(Robledo and Figueras, 1995; Garcia et al., 2009). Similarly, in the
case of M. sydneyi gill stages, epithelial hyperplasia, hypertrophy
and fusion of gill filaments, as well as hemocyte infiltration have
been observed in the infected oysters (Kleeman et al., 2002a).
Parasitic cells of M. chungmuensis have been observed in associ-
ation with hemocyte infiltration around the gonadal follicles in
heavy infections (Ngo et al., 2003). However, no evidence of a clear
impact on the oyster reproductive cycle has been observed (Park
and Chun, 1989). In the case of M. chungmuensis, the impact is
more related to appearance and market acceptance. Infected oys-
ters present nodule-like swellings on their body, which make them
unattractive for consumers (Berthe et al., 2004). At a physiological
level, mechanical blockage of digestive tubules in the host, conse-
quently linked to reduction in feeding capacities, reduction of con-
dition index, reduction of glycogen storage and gametogenesis, has
been recorded in relation to the presence of M. refringens (Robert
et al., 1991; Villalba et al., 1993a; Camacho et al., 1997). In the case
of cellular immune responses, a reduction of phenoloxidase activ-
ity has been related to the presence of M. sydneyi (Peters and
Raftos, 2003), and low levels of heat-shock proteins HSP70 and
stress protein calreticulin accompany infections with M. refringens
(Fuentes et al., 2002). Recently, proteomic maps constructed from
M. sydneyi-infected Sydney rock oysters identified significant tem-
poral differences in the expression of four hemolymph proteins
(p3, p4, p5 and p6) in oysters with sporulating M. sydneyi infections
(Simonian et al., 2009). Furthermore, data from a recent study on
M. sydneyi suggested than Sydney rock oyster hemocytes can
recognize and phagocytose M. sydneyi and that rock oysters resis-
tant to QX disease may be associated with enhanced phagolysoso-
mal activity against M. sydneyi (Kuchel et al., 2010). Cellular
responses and protein dynamics playing a role in the shellfish
immune response need to be better understood.
In nature, host reaction is regulated by different factors: mainly,
host susceptibility and pathogen virulence. Probably, this would
explain the variability in observations regarding host responses,
even prior to Marteilia spp. infection, where we could considerer
all the range of possibilities for both factors, as well as a wide range
of combinations of factors. Knowledge of the complete develop-
mental cycle of the parasite inside the bivalve host would help to
elucidate the role and circumstances associated with those partic-
ular developmental stages and the meaning of unusual pathogen
locations within the host. Similarly, better knowledge of bivalve
45
humoral and cellular defense mechanisms could explain differ-
ences in host susceptibility and differential impacts of Marteilia
spp. parasites in the different affected populations, as would
improved understanding of genotypic differences in parasite viru-
lence. Berthe et al. (2004) pointed out some unpublished data on
how French mussels, with a long historical association with M.
refringens, seemed to find an equilibrium host–parasite relation-
ship, which helped to maintain the parasite prevalence level low
and the associated mortalities reduced or null, in contrast to what
happened to naïve imported mussels introduced in the same area.
Of course, in such an equilibrium, the advantage of bivalve adapta-
tion to local environmental conditions also plays a relevant role in
host susceptibility. This observation is in line with the results
obtained by Fuentes et al. (2002) who studied differential suscep-
tibility to M. refringens by M. galloprovincialis based on the latter’s
genetic origin. In fact, the host–parasite equilibrium is a natural
tendency in nature, which benefits both host and parasite (Roy
and Robert, 1978).
2.2. Developmental stages of Marteilia parasites in bivalve hosts
Paramyxean are protozoan parasites of marine invertebrates
with a characteristic cell-within-cell development occurring by
endogenous budding. Sporogony has also been described for these
parasites, however no other systems of reproduction have been
observed so far. Briefly, M. refringens primary plasmodia-like cells,
with a diameter around 10 lm, are commonly observed in the
epithelium of the stomach of its bivalve host during early develop-
mental stages (Fig. 1). In this stage, multiplication of a primary cell
has been hypothesized to take place before, or just after, producing
the first secondary cell in order to increase the number of primary
cells, and thus the infection intensity (Franc, 1980; Grizel, 1985;
Berthe et al., 2004). However, the process of multiplication itself
has been not observed by microscopical observation. In the case
of M. refringens, M. cochillia and M. granula that primary cell origi-
nally contains a single secondary cell, which later starts internal
division to form up to 8 secondary cells (or presporangia). Those
young stages of the M. refringens parasite (primary cell containing
up to 8 presporangia) seem to migrate to primary and secondary
digestive tubules. The process of migration is not completely
known. While in the primary and secondary digestive tubules, M.
refringens cells continue their maturation process by
cell-within-cell division until they develop mature secondary cells
(sporangia) containing 4 (M. refringens and M. granula) and 6 (M.
cochillia) spores per secondary cell (Grizel et al., 1974; Itoh et al.,
2014; Carrasco et al., 2013a). While M. sydneyi produces up to 16
secondary cells with 2 spores each (Perkins and Wolf, 1976), at
the opposite extreme M. chungmuensis is characterized by having
up to 3 secondary cells containing a single spore each (Comps,
1983). Marteilia-genus mature spores contain 3 sporoplasms
(Feist et al., 2009). Mature spores confined inside sporangia will
be mechanically released into the lumina of the digestive tubules
to be dispersed in the environment via the feces in the case of
digestive-site parasites and via oocytes for the ovarian ones. This
process, in the case of digestive-site parasites, will result in tissue
disruption and the other physiological dysfunctions mentioned
earlier, and potential death of the animal.
3. Geographical distribution and host range
Marteilia spp. have been observed in different invertebrate
hosts in a wide range of geographical locations including Europe,
Oceania, Asia and Africa, but with only a single report from
the Americas (Grizel et al., 1974; Perkins and Wolf, 1976;
46 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
Comps et al., 1986; Moyer et al., 1993; Elgharsalli et al., 2013;
Audemard et al., 2002; Adlard and Nolan, 2015) (Fig. 2; Table 1).
3.1. Marteilia spp. in Europe
3.1.1. M. refringens in oysters, mussels and clams
M. refringens is currently found in several European countries
including France, Spain (Villalba et al., 1992), Italy (Ceschia et al.,
1992), Portugal (Ruano and Dias, 1994), Croatia (Zrnicic et al.,
2001), Greece (Virilis and Angelidis, 2006), Sweden and UK
(see: http://oie.int/wahis_2/public/wahid.php/Diseaseinformation/
statuslist), as well as in Albania (Pëllumb et al., 2006), Slovenia
(Gombac et al., 2014) and a single isolated report from the
Netherlands (Van Banning, 1979). M. refringens infecting
European flat oysters O. edulis was the first Marteilia parasite
described in Europe and around the world (Grizel et al., 1974). It
was found infecting cultured flat oysters in Brittany, France. A
few years later, a Marteilia sp. was also observed in mussels M. edu-
lis imported to France from the Venice Lagoon (Italy). This parasite
was named M. maurini (Comps et al., 1982) based on host affinity
and potential differences observed by transmission electron micro-
scopy (TEM). Later, M. refringens was reported in M. galloprovin-
cialis from Galicia, Spain (Villalba et al., 1992; 1993a).
Ultrastructural TEM and molecular characterization of the internal
transcribed spacer (ITS-1) and intergenic spacer (IGS) regions of
the rRNA gene suggested the existence of a single Marteilia spp.
parasitizing mussels and flat oysters in Europe (Longshaw et al.,
2001; López-Flores et al., 2004): M. refringens, with two main geno-
types, type O and type M (Le Roux et al., 2001), affecting preferen-
tially oysters and mussels, respectively. However, host specify of
those genotypes is not strict, and type O can be found in mussels,
together with type M, and type M can be found in oysters, together
with type O (López-Flores et al., 2004; Novoa et al., 2005; Balseiro
et al., 2007). Thus, they can be found causing cross infections, type
M in oysters and type O in mussels, but more often in co-infection
(both in the same host) in areas where M. refringens-infected
mussels and infected oysters cohabit. However, when
environmental parasite loads are very high, it is probable that
the predominant type infects both oysters and mussels (Carrasco
Fig. 2. Map of Marteilia spp. geographical distribution. Legend: yellow d (Marteilia sp. in oyster species), red N (Marteilia sp. in mussels) and orange j (Marteilia sp. in clams,
cockles and scallops). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
et al., 2007). M. refringens type O has been also detected in C. gallina
from the Balearic Islands (Spain) and M. refringens type M, in Solen
marginatus from Huelva, on the southwest coast of Spain
(López-Flores et al., 2008a, 2008b), as well as type M in the intro-
duced mussel Xenostrobus securis in Galicia, Spain (Pasqual et al.,
2010) (Table 1). Those findings suggest than M. refringens does
not have strict host specificity, but has a relatively wide host range
– parasitizing different bivalve species, as well as different zoo-
planktonic species (see life cycle section).
M. refringens dynamics in O. edulis and mussel populations
seems to be mainly regulated by water temperature. Studies
showed that 17 °C seems to be a trigger for parasite multiplication
and transmission, followed by the disease outbreak (Balouet, 1977;
Grizel, 1985; Audemard et al., 2001). Host mortality seems to occur
when a massive liberation of mature parasite stages mechanically
breaks the digestive tubules causing oyster death. Mortalities of O.
edulis due to M. refringens have reached values up to 100% in some
cases (Berthe et al., 2004), having dramatic socio-economical con-
sequences (e.g. France in the 1970s). In European and North African
waters, the optimal period for parasite development usually occurs
between May and September. However, recent studies revealed
that, at least in Mediterranean waters where temperatures reach
high values, parasites have two periods or cycles, for optimal mul-
tiplication and transmission during the year: spring/summer and
autumn (Boyer et al., 2013). This dynamic seems to correlate with
other biological cycles, including bivalve spawning, zooplankton
community dynamics or other pathogen dynamics in the area,
which are probably linked to food abundance and temperatures.
Other studies recorded mature stages of the parasite during the
entire year (Robledo and Figueras, 1995; Villalba et al., 1993a).
Thus, parasite dynamics in the environment is not completely
understood. It is not known whether these mature stages outside
the known transmission periods are infective and whether trans-
mission can be produced all year around. Thus, the life cycle of
the parasite is not completely known, but an alternative host is
suspected to play an important role (see life cycle section).
Nevertheless, it is suggested that newly exposed oysters become
infected when parasite infective stages are acquired by the host
when filter feeding.
 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57 47

Table 1
Marteilia species and related hosts. Description area and characterization type.

Host Marteilia spp. 1st Reference Origin/ocean Chracterization

Oysters
Ostrea edulis M. refringens Grizel et al. (1974) Europe A/M 18S, IGS, ITS-1, TEM
Ostrea stentina M. refringens Elgharsalli et al. (2013) Tunisia M ITS-1
Ostrea puelchana Marteilia sp. Pascual et al. (1991) France A –
Ostrea angasi Marteilia sp. Bougrier et al. (1986) France A/Australia P –
Ostrea chilensis Marteilia sp. Grizel et al. (1982) France A –
Saccostrea glomerata M. syneyi Perkins and Wolf (1976) Australia P/I 18S, ITS-1, TEM
Saccostrea echinata Marteilioides sp. Hine and Thorne (2000) Australia I –
Saccostrea cucullata Marteilia lenghei Comps et al. (1986) Persian Gulf I TEM
Australia I
Saccostrea forskali Marteilia sp. Taveekijakarn et al. (2002) Thailand P –
Crassostrea gigas Marteilia sp. Cahour (1979) France A –
C. gigas M. chungmuensis Comps et al. (1986) Korea P TEM
C. virginica Marteilia sp. Renault et al. (1995) France A TEM
C. nippona M. chungmuensis Itoh et al. (2004a, 2004b) Japan P 18S
C. ariekensis M. chungmuensis Limpanont et al. (2013) Korea P 18S
Pinctada margaritirefa Marteilia sp. Berthe et al. (2004) Australia –
Mussels
Mytilus galloprovincialis M. refringens Comps and Joly (1980) Europe A/Morocco M IGS and ITS-1
M. edulis Tigé and Rabouin (1976) TEM
Modiolus modiolus Marteilia sp. Auffret and Poder (1987) France A –
Xenostrobus securis M. refringens Pasqual et al. (2010) Spain A IGS
M. edulis Marteilia sp. Wang et al. (2012) China P 18S
Clams/scallops/cockles
Argopecten gibbus Marteilia sp. Moyer et al. (1993) Florida (USA) A –
Scrobicularia piperata Marteilia christienseni Comps (1983) France A TEM
Tapes pullastra Marteilia sp. Poder et al. (1983) France A –
Tapes rhomboides Marteilia sp Poder et al. (1983) France/Spain A –
Villalba et al. (1993a, 1993b)
Ruditapes decussatus Marteilia sp. Villalba et al. (1993a, 1993b) Spain A –
Ruditapes philippinarum Marteilia sp. Itoh et al. (2005) Japan P TEM
Marteilia granula Itoh et al. (2014) Japan P 18S, TEM
Tridacna maxima Marteilia sp. Norton et al. (1993) Fiji P TEM
Ensis minor/E. siliqua Marteilia sp. Ceschia et al. (2001) Italy M –
Solen marginatus Marteilia sp. López and Darriba (2006) Spain A (Galicia) TEM
M. refringens López-Flores et al. (2008b) Spain A (Huelva) IGS
Chamalea gallina M. refringens López-Flores et al. (2008a) Spain M IGS
Cerastoderma edule Marteilia sp. Comps et al. (1975) France A TEM
Marteilia cochillia Carrasco et al. (2013a) Spain M/A IGS, ITS-1, TEM

Legend: A = Atlantic; M = Mediterranean; P = Pacific; I = Indic.

3.1.2. Marteilia christienseni in Scobricularia piperata in France
The species Marteilia christienseni Comps, 1983 was described in
the peppery furrow shell Scobricularia piperata, in Ronce les bains,
on the Atlantic coast of France, based on host affinity and TEM
observations (Comps, 1983). However, since then no information
has been recorded on that species or its host.
3.1.3. Marteilia sp. in oysters, mussels, cockles and clams in Europe
Marteilia spp. parasites have been also observed in different
bivalve species in several European locations (Table 1); however,
the characterization at species level has not been completed in
many of the cases. Thus, Marteilia sp. cells were observed in the
northern horse mussel Modioulus modiolus on the Atlantic coast
of France (Auffret and Poder, 1987), as well as in the clams
Ruditapes pullastra and R. rhomboides in France (Poder et al.,
1983). In this last species, as well as in R. decussatus, the parasite
was also observed in Spanish waters (Villalba et al., 1993b).
Marteilia sp. cells were also recorded in the minor jacknife clam
and pod razor clam, Ensis minor and E. siliqua respectively, in
Italy (Ceschia et al., 2001), as well as in the razor clam Solens
marginatus (López and Darriba, 2006), showing in this last case dif-
ferences with M. refringens taxonomic characters. Also the cockle C.
edule from the Atlantic coast of France was observed to be infected
with a Marteilia sp. (Comps et al., 1975; Poder et al., 1983; Auffret
and Poder, 1987). More recently, the Marteilia sp. observed in cock-
les in Spain has been named as M. cochillia (Carrasco et al., 2013a;
Villalba et al., 2014). Marteilia spp., probably M. refringens, was also
observed in the oysters O. puelchana, O. angasi, O. chilensis during
field experiments in flat oyster O. edulis productions areas in
France where Marteilia-caused disease was enzootic (Grizel et al.,
1982; Bougrier et al., 1986; Pascual et al., 1991). Marteilia sp.
parasites have also been observed in field experiments with the
eastern (American) oyster Crassostrea virginica in France (Renault
et al., 1995) and in C. gigas (Cahour, 1979; Montes et al., 1998)
including, in this last species, the observation of mature stages of
the parasite (S. Darriba, INTECMAR, personal communication).
However, these observations are isolated, and those oyster species
are not currently considered natural hosts of Marteilia parasites.
3.2. Marteilia spp. in Oceania
3.2.1. Marteilia sydneyi from Saccostrea glomerata
Marteilia sydneyi has been identified as the causative agent of
Queensland Unknown (QX) disease of the Sydney rock oyster,
Saccostrea glomerata (Perkins and Wolf, 1976) (Fig. 1). QX disease
causes recurring mass mortalities of S. glomerata in some estuaries
on the east coast of Australia (Green et al., 2011; Nell, 2001).
M. sydneyi usually infects S. glomerata in the southern hemisphere’s
summer months of January to April (Wesche et al., 1999), with
mortality usually occurring between April and June. Infective
stages of M. sydneyi initially enter the oyster via the gills or
labial palps (Kleeman et al., 2002a), usually over a short period
48 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
(sometimes less than 2 wk) during the summer (Adlard and Ernst,
1995). These undergo extrasporogonic proliferation in the gills,
releasing parasitic cells into the surrounding connective tissue
and hemolymph spaces. After systemic dissemination, the parasite
infiltrates the digestive gland and becomes established as a nurse
cell beneath the epithelial cells in a digestive tubule. Here,
cell-within-cell proliferation results in the eventual liberation of
daughter cells from the nurse cell into spaces between adjacent
epithelial cells (Kleeman et al., 2002a). Daughter cells internally
cleave secondary cells, after which degeneration of the nurse cell
occurs. Sporulation proceeds, and mature sporonts containing 2
tricellular spores are shed into the digestive tubule lumen
(Perkins and Wolf, 1976). The majority of sporonts infecting the
digestive gland are shed into the water column by the infected
oysters before they die (Roubal et al., 1989).
3.2.2. Marteilia sp. from Saccostrea cuccullata
Marteilia sp. (probably M. lengehi) was reported infecting the
digestive gland of the Natal rock oyster S. cuccullata in Western
Australia (Hine and Thorne, 2000; Jones and Creeper, 2006). The
parasite resembled M. lengehi reported from the same host in the
Persian Gulf by Comps et al. (1986). If they are conspecific,
M. lengehi or similar species probably occurs across the entire
Indian Ocean, thus linking west and east Eurasia. The prevalence
of the infection detected in Western Australia was low and not
associated with an epizootic. No reports of Marteilia sp. infecting
S. cucullata have since been recorded.
3.2.3. Marteilia sp. from Tridacna maxima and Picnada margaritifera
A single report has been published of a Marteilia sp. infecting
the kidney of the giant clam, Tridacna maxima in Fiji (Norton
et al., 1993). The proliferation of this Marteilia sp. within the kidney
caused large lesions that suggest this parasite is pathogenic in
giant clams (Norton et al., 1993). Berthe et al. (2004) also mention
the presence of a Marteilia sp. parasite in the black-lip pearl oyster
in Australia (Jones in Berthe et al., 2004).
3.2.4. Marteiliodes sp. from Saccostrea echinata
An ovarian parasite, tentatively identified as Marteilioides sp.
was observed in female S. echinata from Darwin Harbour,
Western Australia (Hine and Thorne, 2000; Jones and Creeper,
2006). Following the last classification by Feist et al. (2009),
however, this parasite would belong to the genus Marteilia, thus
Marteilia sp. from S. echinata. Under the light microscope the para-
sites appears to lie within a vacuole and consists of cells within
cells. Sometimes 2 stages were present in a ‘‘69’’ configuration.
Ultrastructurally, endogenous budding within a primary cell pro-
duced a secondary cell, giving rise to 1 or 2 sporangial (tertiary)
cells from each of which one spore develops (Hine and Thorne,
2000). Parasite development did not appear to affect the ova
(Hine and Thorne, 2000).
3.2.5. Other Marteilia-like species
3.2.5.1. Marteiliodes branchialis from Saccostrea glomerata. A single
report describing Marteilioides branchialis infecting the gills of
S. glomerata exists (Anderson and Lester, 1992). The development
of M. branchialis is typical of other paramyxea, whereby a stem cell
internally cleaves a secondary cell contained within a vacuole.
Infection results in focal gill lesions similar in appearance to those
caused by M. sydneyi, but secondary cells of M. branchialis are tri-
cellular compared to M. sydneyi whose secondary cells are bicellu-
lar (Kleeman et al., 2002a). Recently this species has been included
in the genus Paramarteilia, by Feist et al. (2009), thus P. branchialis;
however, its taxonomical position seems to be uncertain.
3.3. Marteilia spp. in Asia
3.3.1. Marteilia chungmuensis from Crassostrea spp.
In addition to the digestive-site species, an ovarian paramyxean
parasite is recognized in oocytes of the Pacific oyster, Crassostrea
gigas. This Marteilia sp. has fewer sporonts (usually only two) in
each sporangium when compared to Marteilia sp. that infect the
digestive gland. Comps et al. (1986) designated this ovarian para-
site as Marteilioides chugmuensis, with creation of a new genus.
Recently, Feist et al. (2009) suggested modification of paramyxean
taxonomy, and this parasite was reclassified into genus Marteilia,
based on the number of cells constructing the spore. Early develop-
mental stages of M. chugmuensis resemble those of Marteilia spp.
that infect the digestive gland; the parasite invades the host
through mantle, gill and labial palps, and then starts multiplication
in connective tissues (Itoh et al., 2004a). Sporulation occurs in
oocytes, and then the parasite cells, retained in oocytes, are
released from the host through the genital canal (Itoh et al.,
2002). So far, direct transmission between C. gigas has not been
established, and the presence of an alternative host(s) is
hypothesized.
Although mortality caused by M. chungmuensis infection is not
high (Tun et al., 2008a, 2008b), affected oysters continuously main-
tain ovaries and produce oocytes after the spawning season, result-
ing in a commercially unacceptable appearance with nodule-like
masses on the ovaries. This disease is referred to as Abnormal
Enlargement of Ovary (AEO), and since its prevalence reaches high
levels (50%) in harvest seasons, AEO is a significant concern for the
Pacific oyster industry in southern part of Korea and western Japan.
So far, mechanisms responsible for unseasonable oogenesis in
affected oysters have not been elucidated, but oogenesis occurs
only in infected follicles and oocytes produced there harbor para-
sites, suggesting that M. chungmuensis may manipulate oyster
reproductive systems to produce oocytes even after spawning sea-
son. The SSU rRNA gene sequence of M. chungmuensis is available,
and recent phylogenetic analysis suggested that this parasite
should be included in the genus Marteilia (Itoh et al., 2014),
although this phylogenetic position is not statistically supported.
SSU rRNA gene sequence information has also been applied to
develop species-specific diagnosis by in situ hybridization and
PCR (Itoh et al., 2003a, 2003b; Choi et al., 2012).
Infection by M. chungmuensis has been also found in the Iwagaki
oyster, C. nippona (Itoh et al., 2005) and the Suminoe oyster,
C. ariakensis (Limpanont et al., 2013), but the nodule-like
appearance on the gonad is not found in these species. This
gonadal abnormality is likely related to host reproductive charac-
teristics, and absence of the nodular masses on these oyster species
may be caused by different reproductive traits. A morphologically
indistinguishable parasite has been reported from oocytes of the
Manila clam, R. philippinarum, in Korea and Japan (Lee et al.,
2001; Itoh et al., 2005), but it is likely that this represents a
different species from M. chungmuensis based on SSU rRNA gene
sequencing.
3.3.2. Marteilia sp. from the rock oyster Saccostrea forskali
A Marteilia sp. infection of rock oysters, Saccostrea forskali, in the
upper part of the Gulf of Thailand was observed by Taveekijakarn
et al. (2008). Sporulation stages with eosinophilic refringent bodies
were found in the epithelium of digestive diverticula, while extra
sporogonic proliferation stages were observed in the gill.
Morphologically, each sporangiosorus (sporangium) contains 2–6
sporonts with two spores, suggesting that this species is morpho-
logically similar to M. sydneyi in a proximate host oyster, S. glomer-
ata. Infection has been found in dry and cold seasons, but the
detection frequency is less than 2%. While hemocyte infiltration
was found around infected loci, no mass mortality associated with
 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
this infection has been reported. Neither DNA sequences nor
detailed ultrastructural information for species identification is
available.
3.3.3. Marteilia sp. from Mytilus edulis
Digestive-site Marteilia sp. in the blue mussel M. edulis has been
also reported in China (Wang et al., 2012). Infected mussels were
found in 5 of 13 examined locations from Bohai to East Sea,
although overall prevalence was only 2.8% (n = 180). Although
morphological information for this species is sparse, a partial SSU
rRNA gene sequence was determined using M. refringens PCR pri-
mers, SS1 and SAS1 (Le Roux et al., 1999) and found to be similar
to, but with some differences from, M. refringens, indicating that
this species is likely to be different from Marteilia spp. found in
blue mussels of Europe (Wang et al., 2012). Wang et al. (2012)
reported infected mussels were emaciated, but that further
examination is needed to more fully understand the parasite’s
pathological effect on the host.
3.3.4. Marteilia sp. from Ruditapes philippinarum
Itoh et al. (2005) found Marteilia sp. infection in the digestive
gland of the Manila clam, R. philippinarum, in Yamaguchi, western
Japan. Sporangia of this species have four sporonts, each of which
contain two spores. Compared with M. granula (Itoh et al., 2014,
see Section 4), this species has fewer eosinophilic granules and
no electron-dense monolayer around the spores, clearly indicating
that this is a different species from M. granula. So far, only one
specimen was recovered, and little additional information is avail-
able. Further examination by molecular and ultrastructual meth-
ods are required for species identification.
3.4. Marteilia sp. from other regions around the world
Marteilia spp. infections have been noted in other geographical
locations, including Kuwait in 1997 (see: http://oie.int/wahis_2/
public/wahid.php/Diseaseinformation/statuslist). In the last dec-
ade, Marteilia spp. parasites have been also observed in
North-African oysters and mussels from Mediterranean waters,
as well as in Ostrea stentina in Tunisia (Elgharsalli et al., 2013),
and O. edulis and M. galloprovincialis in Morocco (http://www.oie.
int/fileadmin/Home/esp/Health_standards/aahm/current/2.4.04_
M_REF.pdf). In both cases, the parasite has been characterized as
M. refringens. On the other site of the Atlantic, a single report of
Marteilia sp. parasites was reported in the calico scallop
Argopecten gibbus in Florida USA (Moyer et al., 1993) (Fig. 2).
Marteilia spp. infections are often readily detected by histology
and North America has had intensive surveillance programs run-
ning for many years. Thus, it seems that Marteilia spp. parasites
are absent in commercially important molluscs from that region.
The sole report, in A. gibbus from Florida, was of mortalities nearing
100%, suggesting a novel pathogen in a naïve host. Such a situation
may occur with the introduction of an exotic pathogen, potentially
associated to shipping.
4. Recent discoveries: Marteilia cochillia (Europe) and Marteilia
granula (Asia)
4.1. Marteilia cochillia
M. cochillia (Fig. 1) has been recently described in the common
European cockle, C. edule, in the Ebro Delta, in Catalonia (NW
Mediterranean, Spain) (Carrasco et al., 2013a, 2013b). The first evi-
dence of M. cochillia was from a C. edule mortality event in Fangar
Bay (August 2008), coinciding with high water temperatures, up to
29 °C (M. Fernández., IRTA, personal communication).
49
Histopathological studies showed the presence of two main
pathologies: signs related to the´ digestive epithelial virosiś (DEV)
(Hine and Wesney, 1997) in 100% of the studied individuals and
Marteilia spp. parasites in 40% (Carrasco et al., 2011), both in the
digestive gland. In situ hybridization using Marteilia
genus-specific probes confirmed that the observed parasite
belonged to the genus Marteilia. Further analysis to clarify the par-
asite’s genetic profile, which focused on the M. refringens ITS-1 (Le
Roux et al., 2001) and IGS (López-Flores et al., 2004) regions,
revealed 86% and 83% of similarity with M. refringens ITS-1 and
IGS, respectively. Such results suggested that the cockle Marteilia
(named type ‘‘C’’) was not M. refringens, but a closely related spe-
cies (Carrasco et al., 2012). The ultrastructural studies supported
that contention.
The currently accepted morphological characteristics used to
distinguish taxonomically among different members of the
Phylum Paramyxea, which involve the number of secondary cells
and spores per secondary cell, were investigated to compare the
studied Marteilia sp. with the previously described ones. The cockle
Marteilia sp. profile (4 secondary cells and 6 spores per secondary
cell) did not coincide with previous descriptions, and led authors to
describe M. cochillia as a new species of parasite in Europe linked to
cockle mortalities (Carrasco et al., 2013a). Villalba et al. (2014)
suggested up to eight secondary cells in a primary cell after
studying M. cochillia smears. Whereas these findings were reported
from the Mediterranean coast of Spain, on the Atlantic coast of
Spain, specifically in the Ria de Arousa (Galicia), mortality of
100% occurred in April–May 2012, which drastically affected the
cockle, C. edule, fishery. The main parasite found was M. cochillia,
with prevalences up to 100% (Villalba et al., 2014). This parasite
continues to cause serious problems for the cockle on natural beds,
and the commercial activity is highly affected. The origin of
the parasite and its paths of dispersion are currently unknown.
In 1975, Comps et al. (1975) had already suggested some
ultrastructural differences between M. refringens and the
Marteilia sp. parasite, potentially M. cochillia, observed in C. edule
from the Rivière d́Auray, in France (Atlantic coast). Marteilia
sp. in C. edule from France was also reported by Poder et al.
(1983), and Auffret and Poder (1987).
In summary, to date M. cochillia has been observed in the Ebro
Delta (Spanish Mediterranean) since August 2008 and in Galicia
(Spanish Atlantic coast) since February 2012, and has, in both sites,
been associated with massive C. edule mortality, when environ-
mental parameters are optimal for parasite proliferate and conse-
quent disease outbreaks (N. Carrasco, personal observation).
Potential commercial connections between producers in these
areas might explain the transfer of this disease agent between
the two affected regions. This connection highlights the extreme
caution required to avoid spreading this parasite to new locations.
Up to now, observations suggest that M. cochillia is highly host spe-
cific. M. cochillia has been found only in the cockle, C. edule, even
when the cockle is known to cohabit with mussels, flat oysters
and other bivalves. Studied mussels and oysters in all cases showed
M. refringens profile. Further, no M. refringens has been observed in
C. edule, even in areas where the parasite is enzootic, suggesting
high host specificity for M. cochillia (Carrasco et al., 2013a;
Villalba et al., 2014). M. cochillia has been also detected in the cope-
pod, A. latisetosa, (Arzul et al., 2014) in the Mediterranean, suggest-
ing this copepod could be potentially involved in its life cycle.
4.2. Marteilia granula
Marteilia granula (Fig. 1) was described from the Manila clam, R.
philippinarum, in Odawa bay, eastern Japan (Itoh et al., 2014).
While early sporulation stages are found in intestine and stomach
50 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
epithelia, late sporulation occurs in the epithelium of digestive
diverticula, producing mature sporangia containing 8 sporonts.
In general, the presence of many large eosinophilic granules
inhibits detailed observations of mature sporangia histologically,
but imprint specimens from digestive tissues allow detailed obser-
vations of sporonts (secondary cells), which contain 4 multicellular
spores. So far, infection outside the digestive diverticula has not
been confirmed. TEM observations revealed that the spores of
M. granula consist of outermost, inner and innermost cells, and that
the developed spore is covered with a characteristic electron-dense
monolayer, which morphologically distinguishes M. granula from
other described Marteilia species. In spite of morphological
similarity, phylogenetic analysis based on the SSU rRNA gene
indicated that M. granula was most distantly related to other
digestive-site Marteilia species. However, these discrepancies
between morphology and phylogeny may be caused by lack of
sequence information from other paramyxean representatives
(such as Paramarteilia or Paramyxa), or by unrecognized, but
critically important morphological structures needed for taxonomy
in this group.
A specific PCR primer pair to detect M. granula has been devel-
oped, and their use in diagnoses appeared to be more sensitive
than conventional histology or tissue imprints of the digestive
gland (Itoh et al., 2014). Mean prevalence of M. granula infection
was less than 10% in 2010–2011 surveys, and not as high as in out-
breaks of M. refringens and M. sydneyi. It seems that infection is
more frequently detected in the winter compared to summer,
although sample numbers for this survey were limited. Because
of low detection frequency and little histopathologically evident
damage, it is unlikely that M. granula has a significant negative
impact on Manila clams.
5. Parasite detection and molecular phylogeny
5.1. Parasite detection
5.1.1. Molecular detection of M. refringens and M. cochillia
Histology and cytology are common techniques used to screen
for the presence of Marteilia spp. parasites in molluscan popula-
tions. The parasite structure (cell-within-cell) is characteristic,
with cell diameters between 5 and 35 lm depending on the devel-
opmental stage, and thus is easily recognizable by microscopical
techniques. However, for corroboration and specific identification,
molecular characterization is required. In recent decades new tools
have been developed for this purpose. These methods allow
detecting and discriminating among Marteilia spp. parasites at
the species level, and in some cases at genotype level. Regarding
Table 2
Molecular detection of Marteilia parasites.
Parasite specie Primer pair/enzyme
*
M. refringens SS2/SAS1
Pr4/Pr5
MT-1/MT-2 and MT-1B/MT-2B
Pr4/Pr5 + Hha I
M. cochillia MT-1/MT-2 and MT-1B/MT-2B + Bgl II
Mcoch-F/Mcoch-F
M. sydneyi LEG1/PRO2
M. chungmuensis MCSP-01 and MCSP-05/6-R
OPF2/OPR2 and OPF3/OPR3
Ish1F/Ish1R
M. granula Para_JP_Af/Para_JP_Dr
Para_JP_Af/Para_JP_Cr
*
This primer pair amplifies and label an 18S region of M. refringens, M. cochillia and M. sydneyi, and probably other Marteilia spp.
the species of Marteilia identified in Europe, molecular methods
to detect M. refringens, such as conventional PCR based on the
small subunit ribosomal RNA gene (Table 2) have been developed
targeting the18S region, with primers SS2/SAS1 (Le Roux et al.,
1999); the internal transcribed spacer (ITS-1) with primers
Pr4/Pr5 (Le Roux et al., 2001); and the intergenic spacer (IGS)
region with a nested PCR using primers MT-1/MT-2 and
MT-1b/MT2b (López-Flores et al., 2004) (Table 1). The conventional
PCR targeting the 18S region is focused on a homogenous sequence
region, less variable than ITS-1 and IGS regions. This PCR assay can
detect M. refringens, M. cochillia and M. sydneyi and probably other
Marteilia spp. It seems to be specific for identifying the genus
Marteilia. Furthermore, the SMART-2 probe based on the 18S
region has been also successfully developed to confirm
Marteilia-genus parasites on histological slides using in situ
hybridization (Le Roux et al., 2001). The ITS-1 PCR assay amplifies
M. refringens type O, type M, and M. cochillia. Furthermore, an
RFLP-PCR with HhaI enzyme on the ITS-1 region (Le Roux et al.,
2001), which shows two different profiles, can be used to distin-
guish type O vs type M and M. cochillia. These last two showed
the same profile using this methodology, which could result in
misidentifications (Carrasco et al., 2012). Finally, the IGS PCR assay
amplifies the three European Marteilia genotypes. The RFLP-PCR on
the IGS with BglII enzyme showed different profiles for M. refrin-
gens and M. cochillia (Carrasco et al., 2012) (Table 2).
Two different real-time PCR assays have also been developed in
order to easily detect M. refringens, minimizing cross contamina-
tion and allowing differentiation between the O and M genotypes
(Carrasco et al., 2013b; I. Arzul, IFREMER, personal communica-
tion). These qPCR assays are currently in process of accreditation/
validation. Other immuno-histochemical procedures with
antibodies were also developed in the past (Tiscar et al., 1993;
Robledo et al., 1994; Berthe et al., 2004), however, they are not
commonly used for research or diagnostics.
5.1.2. Molecular detection of Marteilia sydneyi
Conventional PCR, in-situ hybridisation and immunohistochem-
istry have all been developed for detecting M. sydneyi (Anderson
et al., 1995; Kleeman and Adlard, 2000; Kleeman et al., 2002a,
2002b; Roubal et al., 1989). The conventional PCR and in-situ
hybridisation assays target the internal transcribed spacer 1
(ITS1) region of M. sydneyi (Anderson et al., 1995; Kleeman and
Adlard, 2000). The PCR primers and DNA probe designed for this
region were specific for M. sydneyi when compared with related
Marteilia species (Kleeman et al., 2002b). The sensitivity of the
PCR and in-situ hybridisation assays was assessed using genomic
DNA extracted from a known number of M. sydneyi sporonts. The
Assay Target region Reference
PCR/ISH 18S Le Roux et al. (1999)
PCR ITS-1 Le Roux et al. (2001)
PCR nested IGS López-Flores et al. (2004)
PCR-RFLP ITS-1 + RFLP Le Roux et al. (2001)
PCR-RFLP IGS + RFLP Carrasco et al. (2012)
PCR ITS-1 Villalba et al. (2014)
PCR/ISH ITS-1 Kleeman and Adlard (2000)
ISH 18S Itoh et al. (2003a)
PCR nested 18S Itoh et al. (2003b)
PCR/ISH 18S Choi et al. (2012)
PCR 18S Itoh et al. (2014)
ISH 18S Itoh et al. (2014)
 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
PCR assay could detect the DNA equivalent of 0.01 sporonts follow-
ing agarose gel electrophoresis and the in-situ DNA probe could
detect 10 pg of M. sydneyi DNA in a dot-blot assay (Kleeman and
Adlard, 2000). These molecular tools were primarily developed to
assist in identifying different life stages of M. sydneyi within S.
glomerata (Kleeman et al., 2002a) and for alternative host of
M. sydneyi (Adlard and Nolan, 2015). Nevertheless, histology and
cytology are still routinely used to diagnose farm mortalities of
Sydney rock oysters with QX disease.
5.1.3. Molecular detection of Marteilia granula and Marteilia
chungmuensis
PCR primers, Para_JP_Af and Para_JP_Dr, had higher sensitivity
than histology or tissue imprints for detection of M. granula. PCR
protocol using these probes was highly specific and did not amplify
product from bivalve tissues infected with M. refringens, M. cochil-
lia, M. sydneyi or M. chungmuensis (Itoh et al., 2014). In situ
hybridization probes, Para_JP_Af, Para_JP_Br and Para_JP_Cr, were
also developed to detect M. granula. After further evaluation of
specificity and sensitivity, it is expected that these molecular
probes will be widely utilized for studies on M. granula and its
related species.
Morphologically, developmental stages of M. chungmuensis
outside oyster oocytes have not been well characterized, and
microscopic diagnosis depends on observations for parasite cells
inside oyster oocytes. In order to improve this technical limitation
for diagnosis and studies on the life cycle of the parasite, the18S
rRNA gene sequence of M. chungmuensis was identified and utilized
to develop species-specific nucleotide probes for PCR and in situ
hybridization assays (Itoh et al., 2003a, 2003b). A PCR protocol
using a primer pair, OPF2/OPR2, shows high specificity without
any reaction to related paramyxean parasites. However, sensitivity
of this PCR diagnosis is not sufficient for practical use and
nested-PCR using another primer pair, OPF3/OPR3, is recom-
mended to increase sensitivity (Itoh et al., 2003b). This PCR proto-
col was used for detection of the parasite in male oysters (Itoh
et al., 2004a), diagnosis of various bivalves (Itoh et al., 2004b;
Limpanont et al., 2013; Sühnel et al., 2014) and determination of
invasion period of the parasite (Tun et al., 2008b). In order to mor-
phologically characterize M. chungmuensis cells outside oyster
oocytes, in situ hybridisation probes for M. chungmuensis,
MCSP-01, MCSP-05 and 6-R, were developed (Itoh et al., 2003a).
A combination of an established in situ hybridization protocol
and TEM observations revealed most development stages of
M. chungmuensis inside Pacific oysters (Itoh et al., 2004a). An alter-
native molecular diagnosis for the parasite was developed by Choi
et al. (2012). The PCR protocol developed by these authors does not
require the nested-PCR step, hence may be simpler than the proto-
col developed by Itoh et al. (2003b).
Recently, it has been clarified that an ovarian Marteilia sp. in
Manila clams is phylogenetically different from M. chungmuensis
in oysters, but molecular probes for M. chungmuensis show reactiv-
ity to the clam parasite (Limpanont et al., 2013), hence develop-
ment of molecular probes for each species will be required.
5.2. Molecular phylogeny
The conserved rDNA 18S-coding region has been used to
demonstrate the phylogenetic relationships among M. refringens
and M. cochillia (Europe), M. sydneyi (Australia), and the Asian spe-
cies, M. chungmuensis (Korea, Japan) and M. granula (Japan) (Itoh
et al., 2014). The resulting phylogenetic tree (Fig. 3) shows two
main clusters, one containing the digestive-site Marteilia spp. (M.
refringens, M. cochillia, M. sydneyi and M. granula) and the other
containing the ovarian Marteilia, M. chungmuensis, when using
Maximum-Likelihood method. The first cluster is further divided
51
into three subclusters: European Marteilia spp. (M. refringens and
M. cochillia), M. sydneyi and M. granula. M. refringens and M. cochillia
showed very little differences at the 18S level, with nucleotide
divergence of 2% when the SS2-SAS1 fragment was studied
(Carrasco et al., 2012), while M. granula seem to be consistently
different, at the molecular level, from the other described
Marteilia spp. Further examination of more polymorphic regions
of the genome, such as the ITS-1 and IGS regions, uncovered
genetic differences between closely related species such as, M.
refringens (including type O and M) and M. cochillia (Fig. 3). A
Paramyxean phylogenetic tree including a moderate number of
diverse Paramyxean sequences based on the 18S, ITS-1 and IGS
regions would bring relevant and valuable information on the phy-
logeny of the group. However, to date, the DNA sequence database
for Paramyxean parasites is still weak and more emphasis in this
field of research is required.
6. Advances in life cycle research
6.1. Marteilia refringens
The M. refringens life cycle has been studied since the disease
was described in the 1970s. Replicated parasite transmission stud-
ies between O. edulis oysters, carried out by cohabitation, injection
and feeding of parasite spores has been unsuccessful (Balouet,
1979; Grizel, 1985; Berthe et al., 1998). However, in the case of
mussels, M. galloprovincialis, one successful experiment was
recorded by Comps and Joly (1980) when M. refringens stages from
mashed digestive gland from infected O. edulis apparently infected
‘healthy’ M. galloprovincialis. However, other attempts to directly
transmit the pathogen between bivalves have failed. These early
observations led researchers to suspect a complex life cycle,
involving numerous host species in the transmission of Marteilia
spp. (Balouet, 1979; Grizel, 1985; Lester, 1986; Berthe et al., 2004).
The life cycle of M. refringens and the role of an alternative host
have been extensively studied since the beginning of the new mil-
lennium. These investigations have taken advantage of the
advanced molecular techniques developed during this
time-period. Audemard et al. (2001) studied the M. refringens life
cycle in a French oyster affinage pool, the ‘‘claire’’, looking for
potential alternative hosts. PCR assays showed positive results
for several species: Paracartia grani (Harpaticoid copepod), an
unidentified Cyclopoid copepod, Cereus pendunculatus (Cnidaria),
Lineus gisserensis (Nematoda), among other organisms inhabiting
that confined environment. The limited diversity in the claire made
relatively simple the first step of that complex search (Audemard
et al., 2002). Interest was focused on the copepod Paracartia grani,
which showed by, in situ hybridization, gonadal tissue parasitized
by M. refringens (Audemard et al., 2002). These observations initi-
ated experimental transmission trials that demonstrated M. refrin-
gens could be transmitted from infected O. edulis (through the
feces) to cohabiting P. grani copepods. However, the transmission
from infected P. grani copepods to uninfected O. edulis could not
be demonstrated (Audemard et al., 2002). Later, similar trials were
used to investigate differences in parasite transmission between M.
refringens type ‘‘O’’ (usually found in flat oysters) and M. refringens
type ‘‘M’’ (before M. maurini, mainly found in Mytilus spp. mussels).
These trials revealed that type ‘‘M’’ parasites did not proliferate
after copepod ingestion, while type ‘‘O’’ rapidly proliferated in
infected copepods, migrating from copepod digestive tissue to
the gonad (Carrasco et al., 2008a). Primordia-like (primary cells)
stages similar to the primordial cells described by Adlard and
Nolan (2015) (Fig. 4) were observed by in situ hybridization in
the epithelium of the stomach. Migration stages of the parasite
(nursery cells of Kleeman et al., 2002a), moving from the digestive
52 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57

Fig. 3. Phylogenetical relationships between Marteilia spp. of different geographical origin and host: (A) Phylogenetic relationships of Marteilia species inferred by the
Maximum-Likelihood based on SSU rDNA gene sequences (18S). The Cercozoan Haplosporidium nelsoni was used as the outgroup. Bootstrap values were calculated with 1000
replicates, and values >50% are shown on nodal supports. After Itoh et al., 2014. (B) Neighbor-joining phylogenetical analysis based on MT-1B/MT-2B IGS sequences of
European Marteilia spp. isolates. The hosts for each isolate are indicated by symbols: (N) Mytilus galloprovincialis, (d) Ostrea edulis, (j) Solen marginatus, () Chamalea gallina
and (s) Cerastoderma edule. After Carrasco et al., 2013a.

tissue to the gonad, were also observed by in situ hybridization
(Carrasco et al., 2008a). Observation of semi-thin sections showed
plasmodial forms (20 lm width and 30 lm length), close to a
genital female orifice, containing a large number of Marteilia-like
cells (2-5 lm) at different developmental stages, with the
cell-within-cell arrangements typical of Paramyxeans (Fig. 4).
These resembled the Marteilia-like cells (15 lm) described by
Boyer et al. (2013), also from P. grani. Recent studies showed unu-
sual M. refringens cells in the digestive tract and gonad from the
third copepod stage of P. grani by in situ hybridization during a con-
trolled transmission experiment (Boyer et al., 2013). In this study,
two types of developmental plasmodial stages were observed in
the oocytes: those with very small cells (1–3 lm), which also
resembled to the plasmodial stages described by Adlard and
Nolan (2015), and others with larger Marteilia-like cells (15 lm).
Boyer et al. (2013) suggested that the parasite could infect a cope-
pod by ingestion and be released through the gonad. This hypoth-
esis is supported by the detection of the parasite in copepod eggs
by PCR (Boyer et al., 2013). Further results on mussel retention
efficiency (number and type of copepods retained after filtration),
showed that all copepod developmental stages could contribute
to parasite transmission, especially eggs and nauplii stages
(Boyer et al., 2013).
Life cycle studies at the field level were aimed to enlarge the
‘‘claire model’’ (Audemard et al., 2001) to a wider and more com-
plex natural ecosystem with higher biodiversity than the claire.
The Ebro Delta Alfacs Bay site, was identified as a model location
for the search for alternative hosts, focusing on zooplankton.
Marteilia sp. was detected by PCR in a number of new
zooplanktonic species: several copepods (Acartia discaudata,
A. clausi, A. italica, Oithona sp., Euterpina acutifrons), as well as a
zoeal stage larva of a brachyuran crab (Carrasco et al., 2007).
These results increased the number of potential alternative hosts
for M. refringens, which need to be taken in account for future life
cycle studies (Fig. 4). Further field studies on Marteilia spp. life
cycles carried out in the Diana lagoon, Corsica (France), showed
several additional planktonic taxa that were PCR-positive for
Marteilia spp., including copepods, Cladocera, appendicularia,
 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
Fig. 4. Tentative Marteilia refrigens life cycle and developmental stages in alterna-
tive hosts (modified from Carrasco et al., 2008a).
Chaetognatha and Polychaeta. However, parasite infection could
only be confirmed by in situ hybridization in the copepod
Paracartia latisetosa. Small parasite cells were observed in gonadal
tissues of female P. latisetosa, demonstrating that a copepod
species other than P. grani could be infected by Marteilia spp.
Furthermore, molecular characterization of Marteilia spp. in zoo-
plankton samples from the Diana lagoon allowed three different
genetic profiles for Marteilia sp. to be distinguished: M. refringens
type ‘‘O’’, M. refringens type ‘‘M’’, and Marteilia sp. type ‘‘C’’ (Arzul
et al., 2014), Experimental studies demonstrating the transmission
of Marteilia spp. from infected copepods to uninfected molluscs is
now required in order to corroborate the involvement of copepods
in the life cycle of these parasites.
6.2. Marteilia sydneyi
The first suggestion that M. sydneyi requires more than one host
in its life cycle came over two decades ago with failed attempts to
infect S. glomerata by either cohabitation or transplanting infected
oyster tissue (Lester, 1986; Roubal et al., 1989). In the last decade,
significant progress has been made on identifying an alternative
host in the life cycle of M. sydneyi. Previous research demonstrated
that a benthic and detritivorous invertebrate was likely to be the
alternative host. These conclusion were drawn from research
demonstrating that the majority of M. sydneyi sporonts are shed
into the water column prior to death of S. glomerata and these
sporonts are negatively buoyant (Roubal et al., 1989). Further,
sporonts are short lived (Roubal et al., 1989; Wesche et al.,
1999). Studies demonstrated that sporonts are viable for less than
two hours if ingested by fish or birds (Roubal et al., 1989) and sur-
vive for 9 days in seawater (Wesche et al., 1999). Adlard and Nolan
(2015) sampled the macrobenthos of the Hawkesbury River, New
South Wales (Australia) and found two different developmental
stages of M. sydneyi present in the polychaete worm, Nephtys aus-
traliensis, corroborated by qPCR and in-situ hybridization. A pri-
mordial cell with a well defined nucleus and little differentiation
in the cytoplasm, similar to Carrasco et al. (2008a) for M. refringens
in A. grani, and plasmodial cells with syncytial structure resem-
bling the ‘‘thin’’ stages described by Boyer et al. (2013). Whether
53
this polychaete acts as the secondary host supporting the next
stage in the life cycle of M. sydneyi, or is a ‘‘dead end’’ infection is
yet to be determined. Experimental transmission trials demon-
strating the successful transit of M. sydneyi from S. glomerata to
A. australiensis and vice versa are now warranted.
7. Parasite impact and health management
7.1. M. refringens and M. cochillia socio-economical impacts in
aquaculture and fisheries
The emergence of M. refringens in the 1970s and its economic
and social impact is historically well documented for the French
flat oyster, O. edulis, industry (Grizel et al., 1974). Shortly there-
after, flat oyster farming in other parts of Europe, including
Spain, Italy and Portugal were also devastated by M. refringens
when the disease agent extended to southern latitudes (Villalba
et al., 1992; Ceschia et al., 1992; Ruano and Dias, 1994). In these
countries, aquaculture of O. edulis has been almost completely
replaced by that of the introduced Pacific oyster, C. gigas. The
Pacific oyster did not show pathological problems related to
Marteilia spp. or Bonamia spp. infections as was happening with
O. edulis, and demonstrated a high degree of adaptation to
European waters, with good results in terms of production. The
production of O. edulis in France, as well as in the other European
countries has never recovered to the same production volume as
it was prior to the 1970s outbreaks. Thus, oyster production in
Europe is currently based on the C. gigas industry, which is
currently having important problems associated with the Ostreid
Herpesvirus (OsHv-1 lvar)-caused mass mortality episodes
(Segarra et al., 2010).
Species diversification is a relevant issue in terms of commer-
cial production. Diversification is an important matter for produc-
ers because it helps to widen market strategies and also to mitigate
total commercial losses associated with species-specific patholog-
ical problems.
M. cochillia appears to have a large impact on the production of
the common cockle, C. edule, from beds in Spain (Carrasco et al.,
2013a; Villalba et al., 2014). Prevalences and associated mortalities
have reached 100%, thus causing a collapse of a main economic
resource for Galicia (Atlantic coast of Spain) since 2012. Thus, M.
cochillia has had a significant negative socio-economical impact
for the population of Galicia (Villalba et al., 2014). In Catalonia,
on the Mediterranean coast of Spain, mortality of cockles
associated with M. cochillia have reached 90-100%, also affecting
the cockle fishery in this region. However, proportionally, the
cockle fishery is minor compared to that of Galicia, and
socio-economical consequences are less profound, although still
important for the local sector. In summary, the impact of this par-
asite on the cockle fisheries in Spain is significant. The parasite
needs to be considered of high risk and taken into account by gov-
ernmental authorities since it has not only socio-economical con-
sequences, but also represents a threat to biodiversity because of
the potential disappearance of affected species and the progressive
dispersion of the disease agent.
7.2. Socio-economical impact of Marteilia spp. (including Marteilioides)
in Asia
Pacific oysters infected with M. chungmuensis develop abnor-
mally enlarged ovaries, resulting in a disfigured appearance.
Although infection of the parasite does not cause high mortality
of oysters (Imanaka et al., 2001; Tun et al., 2008a, 2008b), infected
oysters lose marketability due to their appearance. Infection preva-
lence reaches a peak at harvest season, causing serious economical
54 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
loss for oyster industries in Japan. So far, information on econom-
ical loss due to the disease has not been available, but based on
prevalence of the parasite and amount of oyster production, annual
economic loss caused by the disease was estimated to be a few
hundred million Japanese yen (JPY) in the enzootic area,
Okayama (T. Oda, FESOP, personal communication). In addition
to loss of oyster marketability, the parasite may cause spawning
failure of oysters (Ngo et al., 2003); therefore, a decrease in the
oyster resource and failure of artificial spat production are of
concern to the industry. Infection of M. chungmuensis has been
reported in other edible oyster species, such as C. nippona and
C. ariakensis, but prevalences in these species are much lower than
in Pacific oysters and the disfigured appearance has not been
reported (Itoh et al., 2004b; Limpanont et al., 2013), indicating that
M. chungmuensis is not significant threat to the market for these
oyster species.
Although the economical impact of an ovarian Marteilia sp. in
Manila clams has not been documented, it may be negligible,
because of low prevalence and lack of disfigured appearance in
the host. On the other hand, it is believed that clam larvae do not
developed from infected oocytes so that the ovarian Marteilia
sp. may interfere with artificial seed production, which is now
being developed to increase the clam resource in Japan.
The other Marteilia species in Asian waters have been reported
only very recently and little information about their impacts is
available. However, since bivalve production is increasing in Asia,
the pathogenicity and impact of an important parasite group in
the genus Marteilia needs be evaluated as soon as possible.
7.3. Recent advances in management of Martelia spp. parasites with
emphasis on M. sydneyi
Management of bivalve diseases is a complicated matter con-
sidering that they grow in natural environments and thus
chemotherapeutics would be quickly diluted and, additionally,
cannot be used due to their ecological impact. Conventional vacci-
nes used in livestock and finfish production are not effective due to
invertebrates’ lack of adaptive immunity. Thus, the knowledge of
Marteilia spp. parasites’ life cycles could be a key factor to better
control and manage the disease (Audemard et al., 2001).
Currently, the main method of control is the restriction of move-
ments from infected areas to non-infected areas, as well as the har-
vesting or destruction of infected shellfish crops (Berthe et al.,
2004). To our knowledge, few attempts to develop disease resis-
tance to M. refringens by genetic selection have been made to date,
although observations suggest that the biological basis exists
(Fuentes et al., 2002; Berthe et al., 2004). However, considerable
scientific effort (>40 scientific publications) has been invested into
life cycle studies and management of M. sydneyi in an attempt to
mitigate its impact on the Sydney rock oyster industry in the
Australian states of New South Wales (NSW) and Queensland.
Despite this effort, the management options implemented have
failed to prevent the spread of M. sydneyi or to reduce the incidence
of QX disease (reviewed by Green et al., 2011). Management of QX
disease currently relies on a risk-based closure (quarantine) in
NSW (Green et al., 2011), farms diversifying to culturing the exotic
Pacific oyster (Jenkins et al., 2013) and development of
QX-resistant S. glomerata (Dove et al., 2013; O’Connor and Dove,
2009).
The QX-disease risk based closure was implemented by the
NSW Department of Primary Industries in conjunction with the
oyster industry to prevent further spread of QX disease (Green
et al., 2011). Each estuary in NSW has been ranked on its likelihood
of having a QX-disease outbreak (low, medium or high) and oysters
and cultivation equipment can be moved freely between estuaries
within the same risk ranking, and estuaries with lower risk can
move oysters to higher risk estuaries. However, higher risk estuar-
ies cannot move oysters to lower risk estuaries. This approach was
chosen purely because it was the only management tool available
at that time to prevent QX-disease, but the rationale for its imple-
mentation was flawed because QX disease occurred in only a
minority of NSW estuaries, but the disease agent, M. sydneyi, was
present in the majority of NSW estuaries (Adlard and Wesche,
2005). Adlard and Wesche (2005) concluded that QX disease was
caused either by an environmental trigger, an abundance of the
intermediate host in the environment, and/or immunosuppression
of the host. Different strains of M. sydneyi also exist in the river sys-
tems of Southern Queensland and Northern NSW (Kleeman et al.,
2004), but it is not known if they differ in their pathogenicity.
Evidence for genetic variants of M. refringens exists in Europe with
data suggesting a strain more virulent toward O. edulis and another
strain virulent toward Mytilus sp. (Le Roux et al., 2001). Indeed, it
may have been wiser to design the QX-disease risk based closure
around limiting the spread of different M. sydneyi variants between
river systems.
Scientific evidence points to environmental degradation as a
contributing factor in QX disease (Anderson et al., 1994; Diggles,
2013; Green and Barnes, 2010; Wesche, 1995). Historical data sug-
gests that European land-use practices have resulted in sedimenta-
tion of Australian rivers and estuaries resulting in the increased
prevalence of M. sydneyi due to the increased abundance of possi-
ble alternative hosts in the habitat utilized by oysters (Diggles,
2013). Indeed, N. australiensis, the intermediate host is a common
inhabitant of Australia’s east coast estuaries, but is far more abun-
dant in muddy rather than sandy sediments (Hutchings and Rainer,
1979). Immunological studies have shown the immune system of
S. glomerata to be compromised by the presence of some transient
environmental stressor in estuaries enzootic for QX-disease (Peters
and Raftos, 2003; Butt and Raftos, 2007). The transient environ-
mental stressor is likely to be associated with rainwater, or a drop
in salinity, that inhibits the S. glomerata immune system (Butt
et al., 2006). It is therefore not surprising that genetic selection
for QX-resistant S. glomerata has resulted in oysters with elevated
expression levels of genes involved in tolerating environmental
stress, rather than novel immune genes (Green et al., 2009). The
Sydney rock oyster industry is now actively working with local
catchment authorities to inform land users (terrestrial farmers
and local governments) of the importance of maintaining a healthy
river system and the negative impacts on down-stream users, such
as the oyster industry, of not doing so.
Lessons learned from France regarding the management of M.
refringens (Hegaret and Mazurie, 2005) were not heeded in
Australia, with estuaries impacted by reoccurring QX mortality
episodes being granted government approval to grow the
non-native Pacific oyster, C. gigas (Jenkins et al., 2013). As in
France, C. gigas production in NSW originally flourished before
Ostreid herpesvirus type 1 (OsHV-1) devastated the new industry
(Paul-Pont et al., 2014). The development of a QX-resistant S. glom-
erata is showing commercial promise (Dove et al., 2013; O’Connor
and Dove, 2009) and there is renewed interest from oyster growers
to farm the OsHV-1 & QX-disease resistant native flat oyster, Ostrea
angasi.
8. Conclusions and perspectives
Research focused on Marteilia spp. parasites is progressively
contributing to a better understanding of their significance and
impact on bivalve populations around the world. Better knowledge
on Paramyxean phylogeny and potential closely related groups
would help to explain the evolutionary origin of this particular tax-
onomical group of parasites, and would contribute to improved
 N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57
understanding of their biology and life cycles. Molecular advances,
including ‘‘-omics’’ studies and massive sequencing will likely
make a significant contribution in researching the life cycle, para-
site virulence, host susceptibility and host response. These are
issues relevant to reducing the impact of Marteilia-caused disease
by using the most effective management strategies. Experience,
efforts and resources should be co-ordinated and collaboration
encouraged between research teams investigating Paramyxean
parasites. This approach will hopefully fill in the existing knowl-
edge gaps in the near future.
Acknowledgments
NC, would like to thank the INIA postdoctoral contract (Spanish
Government) that has partially covered her professional life
between 2009 and early 2015. Thanks also to the INIA
Complementary Action project 2014/2015, which supported the
1st Paramyxean Working Group meeting in February 2015. The
authors acknowledge funding provided to TG by Macquarie
University Postdoctoral Research Scheme (Grant: 9201300681).
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