Investigation of parasites and diseases affecting molluscs of ecological and economic interest is critical for the management of native stocks and aquaculture. In recent years, much attention has been devoted to investigating the World Organisation for Animal Health listed infectious diseases, so that communities can be prepared to attend public health emergencies and avoid severe income losses. In this context, the health status of Mytilus galloprovincialis Lamarck, 1819 was analysed in two aquaculture sites (Strunjan Bay and Piran Bay, Slovenia), and in four natural mussel beds (Adriatic Croatia International Marinas Pula and Rovinj and St Catherine, Croatia; Marina Koper, Slovenia) along the coast of the northern Adriatic Sea. The mussels were sampled in February and April 2014, and processed for histological examination of several endosymbionts and pathogens that frequently occur in mytilid mussels. Endosymbionts and pathogens were not detected in farmed mussels. Prokaryotic inclusion bodies, the protozoan Nematopsis, Ancistrocoma-like ciliates, haplosporidian-like plasmodia, turbellarian Urastoma cyprinae, and basophilic inclusion bodies were observed in digestive gland cryosections of wild mussels from the coastal region of the northern Adriatic. Fungal spores of Psilocybe sp., Ulocladium sp. and Alternaria sp. were detected between the digestive tubules based on their morphology. Diagnostic PCR did not reveal infection with Marteilia refringens during the studied period, neither in wild nor farmed mussels. We confirmed the site effects on prevalence of infected mussels. Thus, we can conclude that wild areas are more exposed to endobionts and parasites than aquaculture sites.

Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens ‘O-type’ and ‘M-type’. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels (Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within ‘M. refringens’ are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3’ end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

This is the first study of the metabolic enzyme activity and energy state of the dwarf oyster Ostrea stentina (Payraudeau, 1826) in Tunisia and in the Mediterranean region. The main purpose of this study was to examine the modulation of the physiological status of the oyster O. stentina depending on the season and the presence of parasite Marteilia refringens. The prevalence of bonamiosis and marteiliosis were established by PCR. Bonamia exitiosa was detected only in 2.91% of oysters while the prevalence of M. refringens was 100% (30 Ind./30) in February and 93.93% (28 Ind./30) in March. A 42–87% mortality rate was reported during the study period. Biochemical analyses were carried out to evaluate the management of the energy resources in regard to the biochemical changes of lipids and carbohydrates. The enzyme activity of pyruvate kinase (PK), citrate synthase (CS) and cytochrome c oxidase (CCO) were measured and the mean level of enzyme activity was respectively 20.31 mU mg−1 protein; 12.06 mU mg−1 protein and 3.59 mU mg−1 protein. Carbohydrate contents of O. stentina were very low all year round with an average of 15.18% in dry weight, and lipid contents remain similar (11.77% in dry weight) compared with the values reported for most other temperate bivalves. Enzyme activity significantly decreased over time (P < 0.001). The energy resources of the oyster O. stentina wherein much of the energy was devoted to reproduction seem to affect the defence system.

The paramyxean parasite Marteilia refringens infects several bivalve species including European flat oysters Ostrea edulis and Mediterranean mussels Mytilus galloprovincialis. Sequence polymorphism allowed definition of three parasite types ‘M’, ‘O’ and ‘C’ preferably detected in oysters, mussels and cockles respectively. Transmission of the infection from infected bivalves to copepods Paracartia grani could be experimentally achieved but assays from copepods to bivalves failed. In order to contribute to the elucidation of the M. refringens life cycle, the dynamics of the infection was investigated in O. edulis, M. galloprovincialis and zooplankton over one year in Diana lagoon, Corsica (France). Flat oysters appeared non-infected while mussels were infected part of the year, showing highest prevalence in summertime. The parasite was detected by PCR in zooplankton particularly after the peak of prevalence in mussels. Several zooplanktonic groups including copepods, Cladocera, Appendicularia, Chaetognatha and Polychaeta appeared PCR positive. However, only the copepod species Paracartia latisetosa showed positive signal by in situ hybridization. Small parasite cells were observed in gonadal tissues of female copepods demonstrating for the first time that a copepod species other than P. grani can be infected with M. refringens. Molecular characterization of the parasite infecting mussels and zooplankton allowed the distinguishing of three Marteilia types in the lagoon.

Marteilia refringens is a paramyxean parasite which infects the flat oyster Ostrea edulis and mussels (Mytilus galloprovincialis), where it has been attributed to a separate species, Marteilia maurini, by several authors. Doubts persist though as to the existence or not of two species of Marteilia in Europe. We have devised a molecular method for the diagnosis of M. refringens based on 358 bp nested-PCR of the rDNA intergene spacer (rDNA IGS) which is capable of detecting 0·5 fg of M. refringens DNA. Molecular characterization of this spacer indicates that the Marteilia parasites which infect oysters and mussels are two different strains of the same species.

Primers and DNA probes designed for use in the specific detection of the paramyxean parasites Marteilia sydneyi and Marteilia refringens were tested for their potential to cross-react with closely related species in Polymerase Chain Reaction (PCR) and in situ hybridization. PCR primers and a DNA probe designed within the ITS1 rRNA of M. sydneyi were specific for M. sydneyi when compared with related species of Marteilia and Marteilioides. PCR primers designed within the 18S rRNA of M. refringens were specific in the detection of this species in PCR while a DNA probe (named Smart 2) designed on the same gene cross-reacted with M. sydneyi in tissue sections of Saccostrea glomerata as well as Marteilioides sp. infecting Striostrea mytiloides. Though not species specific, the Smart 2 probe provided a stronger signal in detection of all stages of M. sydneyi than the ITS1 probe. The ITS probe is proposed for use as a confirmatory diagnostic tool for M. sydneyi.

Marteilia refringens is a major pathogen of the European flat oyster, Ostrea edulis Linnaeus. Since its description, the life-cycle of this protozoan parasite has eluded discovery. Attempts to infect oysters experimentally have been unsuccessful and led to the hypothesis of a complex life-cycle involving several hosts. Knowledge of this life-cycle is of central importance in order to manage oyster disease. However, the exploration of M. refringens life-cycle has been previously limited by the detection tools available and the tremendous number of species to be screened in enzootic areas. In this study, these two restrictions were circumvented by the use of both molecular detection tools and a mesocosm with low biodiversity. Screening of the entire fauna of the pond for M. refringens DNA was systematically undertaken using PCR. Here, we show that the copepod Paracartia(Acartia) grani is a host of M. refringens. Not only was DNA of M. refringens consistently detected in P. grani but also the presence of the parasite in the ovarian tissues was demonstrated using in situ hybridization. Finally, successful experimental transmissions provided evidence that P. grani can be infected from infected flat oysters.