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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
IRTA-Sant Carles de la Ràpita, Ctra. Poblenou Km 5, 43540 Sant Carles de la Ràpita, Tarragona, Spain
Department of Biological Sciences, Macquarie University, New South Wales 2109, Australia
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,
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 and Wolf, 1976), were thus included in the list of the OIE (World
Organisation for Animal Health) notifiable parasites, and carefully
Marteilia spp. molluscan parasites are protozoans belonging to legislated in the different affected geographical areas (Europe and
the phylum, Cercozoa (Cavalier-Smith, 1998; Cavalier-Smith and Australia) (Anonymous, 2014) in order to be controlled, managed
Chao, 2003), order Paramyxida (Chatton et al., 1911) and character- and their transfer prevented.
ized by their particular cell-inside-cell development. The first Since first detected, M. refringens and M. sydneyi have been stud-
description of a Marteilia sp. parasite corresponded to Marteilia ied using different scientific approaches and new knowledge has
refringens (Grizel et al., 1974) and was associated with mortalities been provided on their development, environmental dynamics
in the European flat oyster Ostrea edulis cultivated in Brittany, and life cycles. However, their complete life cycles are still
France, at the end of the 1960s and early 1970s (Comps, 1970; unknown, becoming one of the more ambitious milestones in this
Herrbach, 1971). Those mortality episodes had a significant impact research field. Recently, studies on the M. refringens life cycle have
on French shellfish aquaculture in terms of socio-economics, pointed out a number of zooplanktonic species, including Acartia
resulting in an important decline of the flat oyster aquaculture in grani and A. latisetosa, which could potentially act as an alternative
Europe, which has never recovered. During a 10-year period host in the life cycle of M. refringens (Audemard et al., 2002;
between the end of the 1960s and beginning of the 1980s, the Carrasco et al., 2007; Boyer et al., 2013; Arzul et al., 2014).
production of flat oysters in France decreased from 20,000 metric Similarly, the polychaete Nephtys australiensis is being studied as
tonnes per year to 2000, in Alderman (1979). The mortality a potential alternative host in the life cycle of M. sydneyi (Adlard
episodes were attributed to the action of two different protozoan and Nolan, 2015). Even with recent advances, many knowledge
parasites: M. refringens and Bonamia ostreae (Berthe et al., 2004). gaps remain regarding the complex life cycles and transmission
Because of the socio-economical impact of those diseases, the two of these parasites. Paramyxean phylogeny, biotype host range
parasites, as well as Marteilia sydneyi, which causes QX disease in and geographical distribution are also important points to be clar-
the Sydney rock oyster, Saccostrea glomerata, in Australia (Perkins ified in order to better understand the impact and associated risks
related with Marteilia spp. parasites.
⇑ Corresponding author at: IRTA, Sant Carles de la Ràpita, Ctra. Poblenou Km 5, The use of molecular tools allowing easy and quick detection as
AP. 200, 43540 Tarragona, Spain. well as parasite molecular characterization has facilitated, in recent
E-mail address: noelia.carrasco@irta.es (N. Carrasco).

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 2. Marteilia spp. parasite development and host response in
Marteilia spp. in new geographical areas, such as China (Wang bivalves
et al., 2012); (2) parasite characterization in new hosts such as
the razor clam Solen marginatus, the Venus clam Chamalea gallina, 2.1. Tissue affinity and host response
the black-pigmy mussel Xenostrobus secures and the common
European cockle Cerastoderma edule (López-Flores et al., 2008a, Marteilia spp. molluscan parasites mainly affect the digestive
2008b; Pasqual et al., 2010; Carrasco et al., 2012, 2013a); and (3) gland, as in the case of M. refringens, M. sydneyi, M. cochillia, and
the description of new Marteilia spp. such as M. cochillia (Carrasco M. granula, or the gonadal tissue, as in the case of M. chungmuensis,
et al., 2013a) in common cockle in Spain and M. granula in the of infected bivalves and other marine invertebrates (Grizel et al.,
Manila clam Ruditapes philippinarum in Japan (Itoh et al., 2014), 1974; Perkins and Wolf, 1976; Audemard et al., 2002; Carrasco
among other findings. Thus, there are many new developments et al., 2013a; Itoh et al., 2014; Adlard and Nolan, 2015) (Fig. 1).
since the last review on Marteilia spp. molluscan parasites (Berthe However, early developmental stages of Marteilia spp. parasites
et al., 2004). The aim of the present review is to summarize have been also described in other tissues. Extrasporogonic early
the new knowledge obtained in the last 10 years, and to stages of M. sydneyi occur in gills (Kleeman et al., 2002a; Green
highlight the priorities for future research. Furthermore, we et al., 2011). Early stages of M. chungmuensis are observed in the
point out that The Paramyxean Working Group Experts Network mantle, gills and labial palps, while multiplication occurs in con-
(www.paramyxeanworkingroup.org), has been also recently nective tissues (Itoh et al., 2004a), and sporulation and mature
created, and it will focus on the advance in this field of research. 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 45

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

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.)
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

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 –
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
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 Villalba et al., 2014). Marteilia spp., probably M. refringens, was also
The species Marteilia christienseni Comps, 1983 was described in observed in the oysters O. puelchana, O. angasi, O. chilensis during
the peppery furrow shell Scobricularia piperata, in Ronce les bains, field experiments in flat oyster O. edulis productions areas in
on the Atlantic coast of France, based on host affinity and TEM France where Marteilia-caused disease was enzootic (Grizel et al.,
observations (Comps, 1983). However, since then no information 1982; Bougrier et al., 1986; Pascual et al., 1991). Marteilia sp.
has been recorded on that species or its host. 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)
3.1.3. Marteilia sp. in oysters, mussels, cockles and clams in Europe
including, in this last species, the observation of mature stages of
Marteilia spp. parasites have been also observed in different
the parasite (S. Darriba, INTECMAR, personal communication).
bivalve species in several European locations (Table 1); however,
However, these observations are isolated, and those oyster species
the characterization at species level has not been completed in
are not currently considered natural hosts of Marteilia parasites.
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 3.2. Marteilia spp. in Oceania
Ruditapes pullastra and R. rhomboides in France (Poder et al.,
1983). In this last species, as well as in R. decussatus, the parasite 3.2.1. Marteilia sydneyi from Saccostrea glomerata
was also observed in Spanish waters (Villalba et al., 1993b). Marteilia sydneyi has been identified as the causative agent of
Marteilia sp. cells were also recorded in the minor jacknife clam Queensland Unknown (QX) disease of the Sydney rock oyster,
and pod razor clam, Ensis minor and E. siliqua respectively, in Saccostrea glomerata (Perkins and Wolf, 1976) (Fig. 1). QX disease
Italy (Ceschia et al., 2001), as well as in the razor clam Solens causes recurring mass mortalities of S. glomerata in some estuaries
marginatus (López and Darriba, 2006), showing in this last case dif- on the east coast of Australia (Green et al., 2011; Nell, 2001).
ferences with M. refringens taxonomic characters. Also the cockle C. M. sydneyi usually infects S. glomerata in the southern hemisphere’s
edule from the Atlantic coast of France was observed to be infected summer months of January to April (Wesche et al., 1999), with
with a Marteilia sp. (Comps et al., 1975; Poder et al., 1983; Auffret mortality usually occurring between April and June. Infective
and Poder, 1987). More recently, the Marteilia sp. observed in cock- stages of M. sydneyi initially enter the oyster via the gills or
les in Spain has been named as M. cochillia (Carrasco et al., 2013a; 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, 3.3. Marteilia spp. in Asia
1995). These undergo extrasporogonic proliferation in the gills,
releasing parasitic cells into the surrounding connective tissue 3.3.1. Marteilia chungmuensis from Crassostrea spp.
and hemolymph spaces. After systemic dissemination, the parasite In addition to the digestive-site species, an ovarian paramyxean
infiltrates the digestive gland and becomes established as a nurse parasite is recognized in oocytes of the Pacific oyster, Crassostrea
cell beneath the epithelial cells in a digestive tubule. Here, gigas. This Marteilia sp. has fewer sporonts (usually only two) in
cell-within-cell proliferation results in the eventual liberation of each sporangium when compared to Marteilia sp. that infect the
daughter cells from the nurse cell into spaces between adjacent digestive gland. Comps et al. (1986) designated this ovarian para-
epithelial cells (Kleeman et al., 2002a). Daughter cells internally site as Marteilioides chugmuensis, with creation of a new genus.
cleave secondary cells, after which degeneration of the nurse cell Recently, Feist et al. (2009) suggested modification of paramyxean
occurs. Sporulation proceeds, and mature sporonts containing 2 taxonomy, and this parasite was reclassified into genus Marteilia,
tricellular spores are shed into the digestive tubule lumen based on the number of cells constructing the spore. Early develop-
(Perkins and Wolf, 1976). The majority of sporonts infecting the mental stages of M. chugmuensis resemble those of Marteilia spp.
digestive gland are shed into the water column by the infected that infect the digestive gland; the parasite invades the host
oysters before they die (Roubal et al., 1989). 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
3.2.2. Marteilia sp. from Saccostrea cuccullata
released from the host through the genital canal (Itoh et al.,
Marteilia sp. (probably M. lengehi) was reported infecting the
2002). So far, direct transmission between C. gigas has not been
digestive gland of the Natal rock oyster S. cuccullata in Western
established, and the presence of an alternative host(s) is
Australia (Hine and Thorne, 2000; Jones and Creeper, 2006). The
parasite resembled M. lengehi reported from the same host in the
Although mortality caused by M. chungmuensis infection is not
Persian Gulf by Comps et al. (1986). If they are conspecific,
high (Tun et al., 2008a, 2008b), affected oysters continuously main-
M. lengehi or similar species probably occurs across the entire
tain ovaries and produce oocytes after the spawning season, result-
Indian Ocean, thus linking west and east Eurasia. The prevalence
ing in a commercially unacceptable appearance with nodule-like
of the infection detected in Western Australia was low and not
masses on the ovaries. This disease is referred to as Abnormal
associated with an epizootic. No reports of Marteilia sp. infecting
Enlargement of Ovary (AEO), and since its prevalence reaches high
S. cucullata have since been recorded.
levels (50%) in harvest seasons, AEO is a significant concern for the
Pacific oyster industry in southern part of Korea and western Japan.
3.2.3. Marteilia sp. from Tridacna maxima and Picnada margaritifera So far, mechanisms responsible for unseasonable oogenesis in
A single report has been published of a Marteilia sp. infecting affected oysters have not been elucidated, but oogenesis occurs
the kidney of the giant clam, Tridacna maxima in Fiji (Norton only in infected follicles and oocytes produced there harbor para-
et al., 1993). The proliferation of this Marteilia sp. within the kidney sites, suggesting that M. chungmuensis may manipulate oyster
caused large lesions that suggest this parasite is pathogenic in reproductive systems to produce oocytes even after spawning sea-
giant clams (Norton et al., 1993). Berthe et al. (2004) also mention son. The SSU rRNA gene sequence of M. chungmuensis is available,
the presence of a Marteilia sp. parasite in the black-lip pearl oyster and recent phylogenetic analysis suggested that this parasite
in Australia (Jones in Berthe et al., 2004). 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
3.2.4. Marteiliodes sp. from Saccostrea echinata
develop species-specific diagnosis by in situ hybridization and
An ovarian parasite, tentatively identified as Marteilioides sp.
PCR (Itoh et al., 2003a, 2003b; Choi et al., 2012).
was observed in female S. echinata from Darwin Harbour,
Infection by M. chungmuensis has been also found in the Iwagaki
Western Australia (Hine and Thorne, 2000; Jones and Creeper,
oyster, C. nippona (Itoh et al., 2005) and the Suminoe oyster,
2006). Following the last classification by Feist et al. (2009),
C. ariakensis (Limpanont et al., 2013), but the nodule-like
however, this parasite would belong to the genus Marteilia, thus
appearance on the gonad is not found in these species. This
Marteilia sp. from S. echinata. Under the light microscope the para-
gonadal abnormality is likely related to host reproductive charac-
sites appears to lie within a vacuole and consists of cells within
teristics, and absence of the nodular masses on these oyster species
cells. Sometimes 2 stages were present in a ‘‘69’’ configuration.
may be caused by different reproductive traits. A morphologically
Ultrastructurally, endogenous budding within a primary cell pro-
indistinguishable parasite has been reported from oocytes of the
duced a secondary cell, giving rise to 1 or 2 sporangial (tertiary)
Manila clam, R. philippinarum, in Korea and Japan (Lee et al.,
cells from each of which one spore develops (Hine and Thorne,
2001; Itoh et al., 2005), but it is likely that this represents a
2000). Parasite development did not appear to affect the ova
different species from M. chungmuensis based on SSU rRNA gene
(Hine and Thorne, 2000).

3.2.5. Other Marteilia-like species 3.3.2. Marteilia sp. from the rock oyster Saccostrea forskali Marteiliodes branchialis from Saccostrea glomerata. A single A Marteilia sp. infection of rock oysters, Saccostrea forskali, in the
report describing Marteilioides branchialis infecting the gills of upper part of the Gulf of Thailand was observed by Taveekijakarn
S. glomerata exists (Anderson and Lester, 1992). The development et al. (2008). Sporulation stages with eosinophilic refringent bodies
of M. branchialis is typical of other paramyxea, whereby a stem cell were found in the epithelium of digestive diverticula, while extra
internally cleaves a secondary cell contained within a vacuole. sporogonic proliferation stages were observed in the gill.
Infection results in focal gill lesions similar in appearance to those Morphologically, each sporangiosorus (sporangium) contains 2–6
caused by M. sydneyi, but secondary cells of M. branchialis are tri- sporonts with two spores, suggesting that this species is morpho-
cellular compared to M. sydneyi whose secondary cells are bicellu- logically similar to M. sydneyi in a proximate host oyster, S. glomer-
lar (Kleeman et al., 2002a). Recently this species has been included ata. Infection has been found in dry and cold seasons, but the
in the genus Paramarteilia, by Feist et al. (2009), thus P. branchialis; detection frequency is less than 2%. While hemocyte infiltration
however, its taxonomical position seems to be uncertain. was found around infected loci, no mass mortality associated with
N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57 49

this infection has been reported. Neither DNA sequences nor Histopathological studies showed the presence of two main
detailed ultrastructural information for species identification is pathologies: signs related to the´ digestive epithelial virosiś (DEV)
available. (Hine and Wesney, 1997) in 100% of the studied individuals and
Marteilia spp. parasites in 40% (Carrasco et al., 2011), both in the
3.3.3. Marteilia sp. from Mytilus edulis digestive gland. In situ hybridization using Marteilia
Digestive-site Marteilia sp. in the blue mussel M. edulis has been genus-specific probes confirmed that the observed parasite
also reported in China (Wang et al., 2012). Infected mussels were belonged to the genus Marteilia. Further analysis to clarify the par-
found in 5 of 13 examined locations from Bohai to East Sea, asite’s genetic profile, which focused on the M. refringens ITS-1 (Le
although overall prevalence was only 2.8% (n = 180). Although Roux et al., 2001) and IGS (López-Flores et al., 2004) regions,
morphological information for this species is sparse, a partial SSU revealed 86% and 83% of similarity with M. refringens ITS-1 and
rRNA gene sequence was determined using M. refringens PCR pri- IGS, respectively. Such results suggested that the cockle Marteilia
mers, SS1 and SAS1 (Le Roux et al., 1999) and found to be similar (named type ‘‘C’’) was not M. refringens, but a closely related spe-
to, but with some differences from, M. refringens, indicating that cies (Carrasco et al., 2012). The ultrastructural studies supported
this species is likely to be different from Marteilia spp. found in that contention.
blue mussels of Europe (Wang et al., 2012). Wang et al. (2012) The currently accepted morphological characteristics used to
reported infected mussels were emaciated, but that further distinguish taxonomically among different members of the
examination is needed to more fully understand the parasite’s Phylum Paramyxea, which involve the number of secondary cells
pathological effect on the host. 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
3.3.4. Marteilia sp. from Ruditapes philippinarum
cell) did not coincide with previous descriptions, and led authors to
Itoh et al. (2005) found Marteilia sp. infection in the digestive
describe M. cochillia as a new species of parasite in Europe linked to
gland of the Manila clam, R. philippinarum, in Yamaguchi, western
cockle mortalities (Carrasco et al., 2013a). Villalba et al. (2014)
Japan. Sporangia of this species have four sporonts, each of which
suggested up to eight secondary cells in a primary cell after
contain two spores. Compared with M. granula (Itoh et al., 2014,
studying M. cochillia smears. Whereas these findings were reported
see Section 4), this species has fewer eosinophilic granules and
from the Mediterranean coast of Spain, on the Atlantic coast of
no electron-dense monolayer around the spores, clearly indicating
Spain, specifically in the Ria de Arousa (Galicia), mortality of
that this is a different species from M. granula. So far, only one
100% occurred in April–May 2012, which drastically affected the
specimen was recovered, and little additional information is avail-
cockle, C. edule, fishery. The main parasite found was M. cochillia,
able. Further examination by molecular and ultrastructual meth-
with prevalences up to 100% (Villalba et al., 2014). This parasite
ods are required for species identification.
continues to cause serious problems for the cockle on natural beds,
and the commercial activity is highly affected. The origin of
3.4. Marteilia sp. from other regions around the world the parasite and its paths of dispersion are currently unknown.
In 1975, Comps et al. (1975) had already suggested some
Marteilia spp. infections have been noted in other geographical ultrastructural differences between M. refringens and the
locations, including Kuwait in 1997 (see: http://oie.int/wahis_2/ Marteilia sp. parasite, potentially M. cochillia, observed in C. edule
public/wahid.php/Diseaseinformation/statuslist). In the last dec- from the Rivière d́Auray, in France (Atlantic coast). Marteilia
ade, Marteilia spp. parasites have been also observed in sp. in C. edule from France was also reported by Poder et al.
North-African oysters and mussels from Mediterranean waters, (1983), and Auffret and Poder (1987).
as well as in Ostrea stentina in Tunisia (Elgharsalli et al., 2013), In summary, to date M. cochillia has been observed in the Ebro
and O. edulis and M. galloprovincialis in Morocco (http://www.oie. Delta (Spanish Mediterranean) since August 2008 and in Galicia
int/fileadmin/Home/esp/Health_standards/aahm/current/2.4.04_ (Spanish Atlantic coast) since February 2012, and has, in both sites,
M_REF.pdf). In both cases, the parasite has been characterized as been associated with massive C. edule mortality, when environ-
M. refringens. On the other site of the Atlantic, a single report of mental parameters are optimal for parasite proliferate and conse-
Marteilia sp. parasites was reported in the calico scallop quent disease outbreaks (N. Carrasco, personal observation).
Argopecten gibbus in Florida USA (Moyer et al., 1993) (Fig. 2). Potential commercial connections between producers in these
Marteilia spp. infections are often readily detected by histology areas might explain the transfer of this disease agent between
and North America has had intensive surveillance programs run- the two affected regions. This connection highlights the extreme
ning for many years. Thus, it seems that Marteilia spp. parasites caution required to avoid spreading this parasite to new locations.
are absent in commercially important molluscs from that region. Up to now, observations suggest that M. cochillia is highly host spe-
The sole report, in A. gibbus from Florida, was of mortalities nearing cific. M. cochillia has been found only in the cockle, C. edule, even
100%, suggesting a novel pathogen in a naïve host. Such a situation when the cockle is known to cohabit with mussels, flat oysters
may occur with the introduction of an exotic pathogen, potentially and other bivalves. Studied mussels and oysters in all cases showed
associated to shipping. M. refringens profile. Further, no M. refringens has been observed in
C. edule, even in areas where the parasite is enzootic, suggesting
4. Recent discoveries: Marteilia cochillia (Europe) and Marteilia high host specificity for M. cochillia (Carrasco et al., 2013a;
granula (Asia) Villalba et al., 2014). M. cochillia has been also detected in the cope-
pod, A. latisetosa, (Arzul et al., 2014) in the Mediterranean, suggest-
4.1. Marteilia cochillia ing this copepod could be potentially involved in its life cycle.

M. cochillia (Fig. 1) has been recently described in the common

European cockle, C. edule, in the Ebro Delta, in Catalonia (NW 4.2. Marteilia granula
Mediterranean, Spain) (Carrasco et al., 2013a, 2013b). The first evi-
dence of M. cochillia was from a C. edule mortality event in Fangar Marteilia granula (Fig. 1) was described from the Manila clam, R.
Bay (August 2008), coinciding with high water temperatures, up to philippinarum, in Odawa bay, eastern Japan (Itoh et al., 2014).
29 °C (M. Fernández., IRTA, personal communication). 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 the species of Marteilia identified in Europe, molecular methods
diverticula, producing mature sporangia containing 8 sporonts. to detect M. refringens, such as conventional PCR based on the
In general, the presence of many large eosinophilic granules small subunit ribosomal RNA gene (Table 2) have been developed
inhibits detailed observations of mature sporangia histologically, targeting the18S region, with primers SS2/SAS1 (Le Roux et al.,
but imprint specimens from digestive tissues allow detailed obser- 1999); the internal transcribed spacer (ITS-1) with primers
vations of sporonts (secondary cells), which contain 4 multicellular Pr4/Pr5 (Le Roux et al., 2001); and the intergenic spacer (IGS)
spores. So far, infection outside the digestive diverticula has not region with a nested PCR using primers MT-1/MT-2 and
been confirmed. TEM observations revealed that the spores of MT-1b/MT2b (López-Flores et al., 2004) (Table 1). The conventional
M. granula consist of outermost, inner and innermost cells, and that PCR targeting the 18S region is focused on a homogenous sequence
the developed spore is covered with a characteristic electron-dense region, less variable than ITS-1 and IGS regions. This PCR assay can
monolayer, which morphologically distinguishes M. granula from detect M. refringens, M. cochillia and M. sydneyi and probably other
other described Marteilia species. In spite of morphological Marteilia spp. It seems to be specific for identifying the genus
similarity, phylogenetic analysis based on the SSU rRNA gene Marteilia. Furthermore, the SMART-2 probe based on the 18S
indicated that M. granula was most distantly related to other region has been also successfully developed to confirm
digestive-site Marteilia species. However, these discrepancies Marteilia-genus parasites on histological slides using in situ
between morphology and phylogeny may be caused by lack of hybridization (Le Roux et al., 2001). The ITS-1 PCR assay amplifies
sequence information from other paramyxean representatives M. refringens type O, type M, and M. cochillia. Furthermore, an
(such as Paramarteilia or Paramyxa), or by unrecognized, but RFLP-PCR with HhaI enzyme on the ITS-1 region (Le Roux et al.,
critically important morphological structures needed for taxonomy 2001), which shows two different profiles, can be used to distin-
in this group. guish type O vs type M and M. cochillia. These last two showed
A specific PCR primer pair to detect M. granula has been devel- the same profile using this methodology, which could result in
oped, and their use in diagnoses appeared to be more sensitive misidentifications (Carrasco et al., 2012). Finally, the IGS PCR assay
than conventional histology or tissue imprints of the digestive amplifies the three European Marteilia genotypes. The RFLP-PCR on
gland (Itoh et al., 2014). Mean prevalence of M. granula infection the IGS with BglII enzyme showed different profiles for M. refrin-
was less than 10% in 2010–2011 surveys, and not as high as in out- gens and M. cochillia (Carrasco et al., 2012) (Table 2).
breaks of M. refringens and M. sydneyi. It seems that infection is Two different real-time PCR assays have also been developed in
more frequently detected in the winter compared to summer, order to easily detect M. refringens, minimizing cross contamina-
although sample numbers for this survey were limited. Because tion and allowing differentiation between the O and M genotypes
of low detection frequency and little histopathologically evident (Carrasco et al., 2013b; I. Arzul, IFREMER, personal communica-
damage, it is unlikely that M. granula has a significant negative tion). These qPCR assays are currently in process of accreditation/
impact on Manila clams. 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
5. Parasite detection and molecular phylogeny commonly used for research or diagnostics.

5.1. Parasite detection

5.1.2. Molecular detection of Marteilia sydneyi
5.1.1. Molecular detection of M. refringens and M. cochillia Conventional PCR, in-situ hybridisation and immunohistochem-
Histology and cytology are common techniques used to screen istry have all been developed for detecting M. sydneyi (Anderson
for the presence of Marteilia spp. parasites in molluscan popula- et al., 1995; Kleeman and Adlard, 2000; Kleeman et al., 2002a,
tions. The parasite structure (cell-within-cell) is characteristic, 2002b; Roubal et al., 1989). The conventional PCR and in-situ
with cell diameters between 5 and 35 lm depending on the devel- hybridisation assays target the internal transcribed spacer 1
opmental stage, and thus is easily recognizable by microscopical (ITS1) region of M. sydneyi (Anderson et al., 1995; Kleeman and
techniques. However, for corroboration and specific identification, Adlard, 2000). The PCR primers and DNA probe designed for this
molecular characterization is required. In recent decades new tools region were specific for M. sydneyi when compared with related
have been developed for this purpose. These methods allow Marteilia species (Kleeman et al., 2002b). The sensitivity of the
detecting and discriminating among Marteilia spp. parasites at PCR and in-situ hybridisation assays was assessed using genomic
the species level, and in some cases at genotype level. Regarding DNA extracted from a known number of M. sydneyi sporonts. The

Table 2
Molecular detection of Marteilia parasites.

Parasite specie Primer pair/enzyme Assay Target region Reference

M. refringens SS2/SAS1 PCR/ISH 18S Le Roux et al. (1999)
Pr4/Pr5 PCR ITS-1 Le Roux et al. (2001)
MT-1/MT-2 and MT-1B/MT-2B PCR nested IGS López-Flores et al. (2004)
Pr4/Pr5 + Hha I PCR-RFLP ITS-1 + RFLP Le Roux et al. (2001)
M. cochillia MT-1/MT-2 and MT-1B/MT-2B + Bgl II PCR-RFLP IGS + RFLP Carrasco et al. (2012)
Mcoch-F/Mcoch-F PCR ITS-1 Villalba et al. (2014)
M. sydneyi LEG1/PRO2 PCR/ISH ITS-1 Kleeman and Adlard (2000)
M. chungmuensis MCSP-01 and MCSP-05/6-R ISH 18S Itoh et al. (2003a)
OPF2/OPR2 and OPF3/OPR3 PCR nested 18S Itoh et al. (2003b)
Ish1F/Ish1R PCR/ISH 18S Choi et al. (2012)
M. granula Para_JP_Af/Para_JP_Dr PCR 18S Itoh et al. (2014)
Para_JP_Af/Para_JP_Cr ISH 18S Itoh et al. (2014)
This primer pair amplifies and label an 18S region of M. refringens, M. cochillia and M. sydneyi, and probably other Marteilia spp.
N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57 51

PCR assay could detect the DNA equivalent of 0.01 sporonts follow- into three subclusters: European Marteilia spp. (M. refringens and
ing agarose gel electrophoresis and the in-situ DNA probe could M. cochillia), M. sydneyi and M. granula. M. refringens and M. cochillia
detect 10 pg of M. sydneyi DNA in a dot-blot assay (Kleeman and showed very little differences at the 18S level, with nucleotide
Adlard, 2000). These molecular tools were primarily developed to divergence of 2% when the SS2-SAS1 fragment was studied
assist in identifying different life stages of M. sydneyi within S. (Carrasco et al., 2012), while M. granula seem to be consistently
glomerata (Kleeman et al., 2002a) and for alternative host of different, at the molecular level, from the other described
M. sydneyi (Adlard and Nolan, 2015). Nevertheless, histology and Marteilia spp. Further examination of more polymorphic regions
cytology are still routinely used to diagnose farm mortalities of of the genome, such as the ITS-1 and IGS regions, uncovered
Sydney rock oysters with QX disease. genetic differences between closely related species such as, M.
refringens (including type O and M) and M. cochillia (Fig. 3). A
5.1.3. Molecular detection of Marteilia granula and Marteilia Paramyxean phylogenetic tree including a moderate number of
chungmuensis diverse Paramyxean sequences based on the 18S, ITS-1 and IGS
PCR primers, Para_JP_Af and Para_JP_Dr, had higher sensitivity regions would bring relevant and valuable information on the phy-
than histology or tissue imprints for detection of M. granula. PCR logeny of the group. However, to date, the DNA sequence database
protocol using these probes was highly specific and did not amplify for Paramyxean parasites is still weak and more emphasis in this
product from bivalve tissues infected with M. refringens, M. cochil- field of research is required.
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 6. Advances in life cycle research
specificity and sensitivity, it is expected that these molecular
probes will be widely utilized for studies on M. granula and its 6.1. Marteilia refringens
related species.
Morphologically, developmental stages of M. chungmuensis The M. refringens life cycle has been studied since the disease
outside oyster oocytes have not been well characterized, and was described in the 1970s. Replicated parasite transmission stud-
microscopic diagnosis depends on observations for parasite cells ies between O. edulis oysters, carried out by cohabitation, injection
inside oyster oocytes. In order to improve this technical limitation and feeding of parasite spores has been unsuccessful (Balouet,
for diagnosis and studies on the life cycle of the parasite, the18S 1979; Grizel, 1985; Berthe et al., 1998). However, in the case of
rRNA gene sequence of M. chungmuensis was identified and utilized mussels, M. galloprovincialis, one successful experiment was
to develop species-specific nucleotide probes for PCR and in situ recorded by Comps and Joly (1980) when M. refringens stages from
hybridization assays (Itoh et al., 2003a, 2003b). A PCR protocol mashed digestive gland from infected O. edulis apparently infected
using a primer pair, OPF2/OPR2, shows high specificity without ‘healthy’ M. galloprovincialis. However, other attempts to directly
any reaction to related paramyxean parasites. However, sensitivity transmit the pathogen between bivalves have failed. These early
of this PCR diagnosis is not sufficient for practical use and observations led researchers to suspect a complex life cycle,
nested-PCR using another primer pair, OPF3/OPR3, is recom- involving numerous host species in the transmission of Marteilia
mended to increase sensitivity (Itoh et al., 2003b). This PCR proto- spp. (Balouet, 1979; Grizel, 1985; Lester, 1986; Berthe et al., 2004).
col was used for detection of the parasite in male oysters (Itoh The life cycle of M. refringens and the role of an alternative host
et al., 2004a), diagnosis of various bivalves (Itoh et al., 2004b; have been extensively studied since the beginning of the new mil-
Limpanont et al., 2013; Sühnel et al., 2014) and determination of lennium. These investigations have taken advantage of the
invasion period of the parasite (Tun et al., 2008b). In order to mor- advanced molecular techniques developed during this
phologically characterize M. chungmuensis cells outside oyster time-period. Audemard et al. (2001) studied the M. refringens life
oocytes, in situ hybridisation probes for M. chungmuensis, cycle in a French oyster affinage pool, the ‘‘claire’’, looking for
MCSP-01, MCSP-05 and 6-R, were developed (Itoh et al., 2003a). potential alternative hosts. PCR assays showed positive results
A combination of an established in situ hybridization protocol for several species: Paracartia grani (Harpaticoid copepod), an
and TEM observations revealed most development stages of unidentified Cyclopoid copepod, Cereus pendunculatus (Cnidaria),
M. chungmuensis inside Pacific oysters (Itoh et al., 2004a). An alter- Lineus gisserensis (Nematoda), among other organisms inhabiting
native molecular diagnosis for the parasite was developed by Choi that confined environment. The limited diversity in the claire made
et al. (2012). The PCR protocol developed by these authors does not relatively simple the first step of that complex search (Audemard
require the nested-PCR step, hence may be simpler than the proto- et al., 2002). Interest was focused on the copepod Paracartia grani,
col developed by Itoh et al. (2003b). which showed by, in situ hybridization, gonadal tissue parasitized
Recently, it has been clarified that an ovarian Marteilia sp. in by M. refringens (Audemard et al., 2002). These observations initi-
Manila clams is phylogenetically different from M. chungmuensis ated experimental transmission trials that demonstrated M. refrin-
in oysters, but molecular probes for M. chungmuensis show reactiv- gens could be transmitted from infected O. edulis (through the
ity to the clam parasite (Limpanont et al., 2013), hence develop- feces) to cohabiting P. grani copepods. However, the transmission
ment of molecular probes for each species will be required. from infected P. grani copepods to uninfected O. edulis could not
be demonstrated (Audemard et al., 2002). Later, similar trials were
5.2. Molecular phylogeny used to investigate differences in parasite transmission between M.
refringens type ‘‘O’’ (usually found in flat oysters) and M. refringens
The conserved rDNA 18S-coding region has been used to type ‘‘M’’ (before M. maurini, mainly found in Mytilus spp. mussels).
demonstrate the phylogenetic relationships among M. refringens These trials revealed that type ‘‘M’’ parasites did not proliferate
and M. cochillia (Europe), M. sydneyi (Australia), and the Asian spe- after copepod ingestion, while type ‘‘O’’ rapidly proliferated in
cies, M. chungmuensis (Korea, Japan) and M. granula (Japan) (Itoh infected copepods, migrating from copepod digestive tissue to
et al., 2014). The resulting phylogenetic tree (Fig. 3) shows two the gonad (Carrasco et al., 2008a). Primordia-like (primary cells)
main clusters, one containing the digestive-site Marteilia spp. (M. stages similar to the primordial cells described by Adlard and
refringens, M. cochillia, M. sydneyi and M. granula) and the other Nolan (2015) (Fig. 4) were observed by in situ hybridization in
containing the ovarian Marteilia, M. chungmuensis, when using the epithelium of the stomach. Migration stages of the parasite
Maximum-Likelihood method. The first cluster is further divided (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 efficiency (number and type of copepods retained after filtration),
(Carrasco et al., 2008a). Observation of semi-thin sections showed showed that all copepod developmental stages could contribute
plasmodial forms (20 lm width and 30 lm length), close to a to parasite transmission, especially eggs and nauplii stages
genital female orifice, containing a large number of Marteilia-like (Boyer et al., 2013).
cells (2-5 lm) at different developmental stages, with the Life cycle studies at the field level were aimed to enlarge the
cell-within-cell arrangements typical of Paramyxeans (Fig. 4). ‘‘claire model’’ (Audemard et al., 2001) to a wider and more com-
These resembled the Marteilia-like cells (15 lm) described by plex natural ecosystem with higher biodiversity than the claire.
Boyer et al. (2013), also from P. grani. Recent studies showed unu- The Ebro Delta Alfacs Bay site, was identified as a model location
sual M. refringens cells in the digestive tract and gonad from the for the search for alternative hosts, focusing on zooplankton.
third copepod stage of P. grani by in situ hybridization during a con- Marteilia sp. was detected by PCR in a number of new
trolled transmission experiment (Boyer et al., 2013). In this study, zooplanktonic species: several copepods (Acartia discaudata,
two types of developmental plasmodial stages were observed in A. clausi, A. italica, Oithona sp., Euterpina acutifrons), as well as a
the oocytes: those with very small cells (1–3 lm), which also zoeal stage larva of a brachyuran crab (Carrasco et al., 2007).
resembled to the plasmodial stages described by Adlard and These results increased the number of potential alternative hosts
Nolan (2015), and others with larger Marteilia-like cells (15 lm). for M. refringens, which need to be taken in account for future life
Boyer et al. (2013) suggested that the parasite could infect a cope- cycle studies (Fig. 4). Further field studies on Marteilia spp. life
pod by ingestion and be released through the gonad. This hypoth- cycles carried out in the Diana lagoon, Corsica (France), showed
esis is supported by the detection of the parasite in copepod eggs several additional planktonic taxa that were PCR-positive for
by PCR (Boyer et al., 2013). Further results on mussel retention Marteilia spp., including copepods, Cladocera, appendicularia,
N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57 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
Fig. 4. Tentative Marteilia refrigens life cycle and developmental stages in alterna-
European waters, with good results in terms of production. The
tive hosts (modified from Carrasco et al., 2008a).
production of O. edulis in France, as well as in the other European
countries has never recovered to the same production volume as
Chaetognatha and Polychaeta. However, parasite infection could it was prior to the 1970s outbreaks. Thus, oyster production in
only be confirmed by in situ hybridization in the copepod Europe is currently based on the C. gigas industry, which is
Paracartia latisetosa. Small parasite cells were observed in gonadal currently having important problems associated with the Ostreid
tissues of female P. latisetosa, demonstrating that a copepod Herpesvirus (OsHv-1 lvar)-caused mass mortality episodes
species other than P. grani could be infected by Marteilia spp. (Segarra et al., 2010).
Furthermore, molecular characterization of Marteilia spp. in zoo- Species diversification is a relevant issue in terms of commer-
plankton samples from the Diana lagoon allowed three different cial production. Diversification is an important matter for produc-
genetic profiles for Marteilia sp. to be distinguished: M. refringens ers because it helps to widen market strategies and also to mitigate
type ‘‘O’’, M. refringens type ‘‘M’’, and Marteilia sp. type ‘‘C’’ (Arzul total commercial losses associated with species-specific patholog-
et al., 2014), Experimental studies demonstrating the transmission ical problems.
of Marteilia spp. from infected copepods to uninfected molluscs is M. cochillia appears to have a large impact on the production of
now required in order to corroborate the involvement of copepods the common cockle, C. edule, from beds in Spain (Carrasco et al.,
in the life cycle of these parasites. 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.
6.2. Marteilia sydneyi
cochillia has had a significant negative socio-economical impact
for the population of Galicia (Villalba et al., 2014). In Catalonia,
The first suggestion that M. sydneyi requires more than one host
on the Mediterranean coast of Spain, mortality of cockles
in its life cycle came over two decades ago with failed attempts to
associated with M. cochillia have reached 90-100%, also affecting
infect S. glomerata by either cohabitation or transplanting infected
the cockle fishery in this region. However, proportionally, the
oyster tissue (Lester, 1986; Roubal et al., 1989). In the last decade,
cockle fishery is minor compared to that of Galicia, and
significant progress has been made on identifying an alternative
socio-economical consequences are less profound, although still
host in the life cycle of M. sydneyi. Previous research demonstrated
important for the local sector. In summary, the impact of this par-
that a benthic and detritivorous invertebrate was likely to be the
asite on the cockle fisheries in Spain is significant. The parasite
alternative host. These conclusion were drawn from research
needs to be considered of high risk and taken into account by gov-
demonstrating that the majority of M. sydneyi sporonts are shed
ernmental authorities since it has not only socio-economical con-
into the water column prior to death of S. glomerata and these
sequences, but also represents a threat to biodiversity because of
sporonts are negatively buoyant (Roubal et al., 1989). Further,
the potential disappearance of affected species and the progressive
sporonts are short lived (Roubal et al., 1989; Wesche et al.,
dispersion of the disease agent.
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 7.2. Socio-economical impact of Marteilia spp. (including Marteilioides)
(2015) sampled the macrobenthos of the Hawkesbury River, New in Asia
South Wales (Australia) and found two different developmental
stages of M. sydneyi present in the polychaete worm, Nephtys aus- Pacific oysters infected with M. chungmuensis develop abnor-
traliensis, corroborated by qPCR and in-situ hybridization. A pri- mally enlarged ovaries, resulting in a disfigured appearance.
mordial cell with a well defined nucleus and little differentiation Although infection of the parasite does not cause high mortality
in the cytoplasm, similar to Carrasco et al. (2008a) for M. refringens of oysters (Imanaka et al., 2001; Tun et al., 2008a, 2008b), infected
in A. grani, and plasmodial cells with syncytial structure resem- oysters lose marketability due to their appearance. Infection preva-
bling the ‘‘thin’’ stages described by Boyer et al. (2013). Whether 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- move oysters to higher risk estuaries. However, higher risk estuar-
ical loss due to the disease has not been available, but based on ies cannot move oysters to lower risk estuaries. This approach was
prevalence of the parasite and amount of oyster production, annual chosen purely because it was the only management tool available
economic loss caused by the disease was estimated to be a few at that time to prevent QX-disease, but the rationale for its imple-
hundred million Japanese yen (JPY) in the enzootic area, mentation was flawed because QX disease occurred in only a
Okayama (T. Oda, FESOP, personal communication). In addition minority of NSW estuaries, but the disease agent, M. sydneyi, was
to loss of oyster marketability, the parasite may cause spawning present in the majority of NSW estuaries (Adlard and Wesche,
failure of oysters (Ngo et al., 2003); therefore, a decrease in the 2005). Adlard and Wesche (2005) concluded that QX disease was
oyster resource and failure of artificial spat production are of caused either by an environmental trigger, an abundance of the
concern to the industry. Infection of M. chungmuensis has been intermediate host in the environment, and/or immunosuppression
reported in other edible oyster species, such as C. nippona and of the host. Different strains of M. sydneyi also exist in the river sys-
C. ariakensis, but prevalences in these species are much lower than tems of Southern Queensland and Northern NSW (Kleeman et al.,
in Pacific oysters and the disfigured appearance has not been 2004), but it is not known if they differ in their pathogenicity.
reported (Itoh et al., 2004b; Limpanont et al., 2013), indicating that Evidence for genetic variants of M. refringens exists in Europe with
M. chungmuensis is not significant threat to the market for these data suggesting a strain more virulent toward O. edulis and another
oyster species. strain virulent toward Mytilus sp. (Le Roux et al., 2001). Indeed, it
Although the economical impact of an ovarian Marteilia sp. in may have been wiser to design the QX-disease risk based closure
Manila clams has not been documented, it may be negligible, around limiting the spread of different M. sydneyi variants between
because of low prevalence and lack of disfigured appearance in river systems.
the host. On the other hand, it is believed that clam larvae do not Scientific evidence points to environmental degradation as a
developed from infected oocytes so that the ovarian Marteilia contributing factor in QX disease (Anderson et al., 1994; Diggles,
sp. may interfere with artificial seed production, which is now 2013; Green and Barnes, 2010; Wesche, 1995). Historical data sug-
being developed to increase the clam resource in Japan. gests that European land-use practices have resulted in sedimenta-
The other Marteilia species in Asian waters have been reported tion of Australian rivers and estuaries resulting in the increased
only very recently and little information about their impacts is prevalence of M. sydneyi due to the increased abundance of possi-
available. However, since bivalve production is increasing in Asia, ble alternative hosts in the habitat utilized by oysters (Diggles,
the pathogenicity and impact of an important parasite group in 2013). Indeed, N. australiensis, the intermediate host is a common
the genus Marteilia needs be evaluated as soon as possible. inhabitant of Australia’s east coast estuaries, but is far more abun-
dant in muddy rather than sandy sediments (Hutchings and Rainer,
7.3. Recent advances in management of Martelia spp. parasites with 1979). Immunological studies have shown the immune system of
emphasis on M. sydneyi S. glomerata to be compromised by the presence of some transient
environmental stressor in estuaries enzootic for QX-disease (Peters
Management of bivalve diseases is a complicated matter con- and Raftos, 2003; Butt and Raftos, 2007). The transient environ-
sidering that they grow in natural environments and thus mental stressor is likely to be associated with rainwater, or a drop
chemotherapeutics would be quickly diluted and, additionally, in salinity, that inhibits the S. glomerata immune system (Butt
cannot be used due to their ecological impact. Conventional vacci- et al., 2006). It is therefore not surprising that genetic selection
nes used in livestock and finfish production are not effective due to for QX-resistant S. glomerata has resulted in oysters with elevated
invertebrates’ lack of adaptive immunity. Thus, the knowledge of expression levels of genes involved in tolerating environmental
Marteilia spp. parasites’ life cycles could be a key factor to better stress, rather than novel immune genes (Green et al., 2009). The
control and manage the disease (Audemard et al., 2001). Sydney rock oyster industry is now actively working with local
Currently, the main method of control is the restriction of move- catchment authorities to inform land users (terrestrial farmers
ments from infected areas to non-infected areas, as well as the har- and local governments) of the importance of maintaining a healthy
vesting or destruction of infected shellfish crops (Berthe et al., river system and the negative impacts on down-stream users, such
2004). To our knowledge, few attempts to develop disease resis- as the oyster industry, of not doing so.
tance to M. refringens by genetic selection have been made to date, Lessons learned from France regarding the management of M.
although observations suggest that the biological basis exists refringens (Hegaret and Mazurie, 2005) were not heeded in
(Fuentes et al., 2002; Berthe et al., 2004). However, considerable Australia, with estuaries impacted by reoccurring QX mortality
scientific effort (>40 scientific publications) has been invested into episodes being granted government approval to grow the
life cycle studies and management of M. sydneyi in an attempt to non-native Pacific oyster, C. gigas (Jenkins et al., 2013). As in
mitigate its impact on the Sydney rock oyster industry in the France, C. gigas production in NSW originally flourished before
Australian states of New South Wales (NSW) and Queensland. Ostreid herpesvirus type 1 (OsHV-1) devastated the new industry
Despite this effort, the management options implemented have (Paul-Pont et al., 2014). The development of a QX-resistant S. glom-
failed to prevent the spread of M. sydneyi or to reduce the incidence erata is showing commercial promise (Dove et al., 2013; O’Connor
of QX disease (reviewed by Green et al., 2011). Management of QX and Dove, 2009) and there is renewed interest from oyster growers
disease currently relies on a risk-based closure (quarantine) in to farm the OsHV-1 & QX-disease resistant native flat oyster, Ostrea
NSW (Green et al., 2011), farms diversifying to culturing the exotic angasi.
Pacific oyster (Jenkins et al., 2013) and development of
QX-resistant S. glomerata (Dove et al., 2013; O’Connor and Dove,
2009). 8. Conclusions and perspectives
The QX-disease risk based closure was implemented by the
NSW Department of Primary Industries in conjunction with the Research focused on Marteilia spp. parasites is progressively
oyster industry to prevent further spread of QX disease (Green contributing to a better understanding of their significance and
et al., 2011). Each estuary in NSW has been ranked on its likelihood impact on bivalve populations around the world. Better knowledge
of having a QX-disease outbreak (low, medium or high) and oysters on Paramyxean phylogeny and potential closely related groups
and cultivation equipment can be moved freely between estuaries would help to explain the evolutionary origin of this particular tax-
within the same risk ranking, and estuaries with lower risk can onomical group of parasites, and would contribute to improved
N. Carrasco et al. / Journal of Invertebrate Pathology 131 (2015) 43–57 55

understanding of their biology and life cycles. Molecular advances, Butt, D., Raftos, D.A., 2007. Immunosuppression in Sydney rock oysters (Saccostrea
glomerata) and QX disease in the Hawkesbury River, Sydney. Mar. Freshwater
including ‘‘-omics’’ studies and massive sequencing will likely
Res. 58, 213–221.
make a significant contribution in researching the life cycle, para- Butt, D., Shaddick, K., Raftos, D.A., 2006. The effect of low salinity on phenoloxidase
site virulence, host susceptibility and host response. These are activity in the Sydney rock oyster, Saccostrea glomerata. Aquaculture 251, 159–
issues relevant to reducing the impact of Marteilia-caused disease 166.
Cahour, A., 1979. Marteilia refringens and Crassostrea gigas. Mar. Fish Rev. 41, 19–20.
by using the most effective management strategies. Experience, Camacho, A., Villalba, A., Beiras, R., Labarta, U., 1997. Absorption efficiency and
efforts and resources should be co-ordinated and collaboration condition of cultured mussels (Mytilus edulis galloprovincialis LINNAEUS) of
encouraged between research teams investigating Paramyxean Galicia (NW Spain) infected by parasites Marteilia refrigens (Grizel et al.) and
Myticola intestinalis (Steven). J. Shellfish Res. 16, 77–82.
parasites. This approach will hopefully fill in the existing knowl- Carrasco, N., López-Flores, I., Alcaraz, M., Furones, M.D., Berthe, F., Arzul, I., 2007.
edge gaps in the near future. Dynamics of the parasite Marteilia refringens (Paramyxea) in Mytilus
galloprovincialisand zooplankton populations in Alfacs Bay (Catalonia, Spain).
Parasitol 134–11, 1541–1550.
Acknowledgments Carrasco, N., Arzul, I., Chollet, B., Robert, M., Joly, J.-P., Furones, M.D., Berthe, F.C.J.,
2008a. Comparative experimental infection of the copepod Paracarti agrani with
Marteilia refringens and M. maurini. J. Fish Dis. 31, 497–504.
NC, would like to thank the INIA postdoctoral contract (Spanish Carrasco, N., Arzul, I., Berthe, F.C.J., Furones, M.D., 2008b. In situ hybridization
Government) that has partially covered her professional life detection of Marteilia refringens (Paramyxea) initial infective stages in its host
Mytilu sgalloprovincialis. J. Fish Dis. 31 (2), 153–157.
between 2009 and early 2015. Thanks also to the INIA Carrasco, N., Roque, A., Andree, K.B., Rodgers, C., Lacuesta, B., Furones, M.D., 2011. A
Complementary Action project 2014/2015, which supported the Marteilia parasite and digestive epithelial virosis lesions observed during a
1st Paramyxean Working Group meeting in February 2015. The common edible cockle Cerastoderma edule mortality event in the Spanish
Mediterranean coast. Aquaculture 321, 197–202.
authors acknowledge funding provided to TG by Macquarie Carrasco, N., Andree, K.B., Roque, A., Rodgers, C., Lacuesta, B., Furones, M.D., 2012.
University Postdoctoral Research Scheme (Grant: 9201300681). Molecular characterization of the Marteilia parasite infecting the common
edible cockle Cerastoderma edule in the Spanish Mediterranean coast. A new
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