Seasonal patterns of metabolism and the heat shock response (HSR) in farmed mussels Mytilus galloprovincialis

Mussels (Mytilus galloprovincialis) are the main bivalves cultured in the Mediterranean Sea and in the Thermaikos gulf North of Greece. Global warming might affect these cultures since increases in the sea temperatures especially during summertime and may cause mass mortalities. Thus projections of the effects of global warming and the knowledge of the mechanisms of thermal limitation and adaptation are considered important in this context. In the present work we studied the seasonal expression of Hsp70 and Hsp90 of aquacultured mussels in nature in an effort to examine whether mussels are seasonally thermally stressed. Moreover we determined the activity of the key glycolytic enzyme PK in order to examine seasonal changes in the metabolic profile. The present data showed a biphasic seasonal pattern in the expression of Hsps in the mantle and PAM of M. galloprovincialis. The expression of inducible Hsp72 and Hsp90 increased between February and mid April, and remained constant or decreased slightly by the end of May. Hsps expression started again in early June leading to a gradual increase by mid July and remained by the end of August. The pattern of the HSR indicates increased protein turnover or protein damage in the tissues of aquacultured M. galloprovincialis between winter and summer seasons. The induction temperature is probably limited, and beyond some upper limits there is no further change in the HSR induction temperatures and the HSR sets in. PK activity in the mantle was significantly higher in winter than in spring. Early May saw a reactivation of PK to higher than spring values. Similar to mantle PK, PK activity in PAM was significantly higher in winter than in spring. A slight reactivation was observed by the end of August.     

Interactive effects of food availability and aerial body temperature on the survival of two intertidal Mytilus species

Intertidal organisms must episodically contend with the rigors of both the terrestrial and the marine environments. While body temperatures during high tide are driven primarily by water temperature, aerial body temperatures are driven by multiple environmental factors such that temperature of an organism during low tide is usually quite different from air temperature. Thus, whereas decades of research have investigated the effects of water temperature on intertidal species, considerably less is known about the physiological impacts of temperature during aerial exposure at low tide, especially with regard to the interaction of aerial body temperature with other stressors. We examined the interactive effects of aerial body temperature and food supply on the survival of two intertidal blue mussels, Mytilus galloprovincialis and Mytilus trossulus. Survival was monitored for nine weeks using a simulated tidal cycle, with two levels of food and three levels of aerial body temperature (30, 25, and 20 °C). Decreased food supply significantly reduced the survival of mussels, but only under the 30 °C treatment. In the other two thermal regimes there were no significant effect of food on survival. When aerial body temperatures are high, food availability may have a greater effect on intertidal organisms. Decreases in ocean productivity have been linked to increased in ocean temperatures, thus intertidal organisms may become more susceptible to thermal stress as climates shift.     

Coccomyxa sp. (Chlorophyta: Chlorococcales), a new pathogen in mussels (Mytilus galloprovincialis) of Vigo estuary (Galicia, NW Spain)

In this work, we describe the occurrence of irregular shaped green aggregations in the mantle, gill filaments, adductor muscle, visceral mass and haemolymph of wild mussels (Mytilus galloprovincialis) collected from the Vigo estuary (Galicia, NW Spain). Microscopic examination of these masses revealed that they consist of intracellular green algae which are spherical to oval in shape, 5 μm in length and 3 μm in width, without flagella and with a smooth surface. The algal cells present a small single nucleus, a mitochondrion, 1-2 parietal chloroplasts and lack pyrenoids. Reproduction is by formation of 2-4 autospores or daughter cells. Pigment analysis reveals the presence of photopigments typical of green algae in addition to alloxanthin, diadinoxanthin and diatoxanthin. These carotenoids are noted for the first time in a parasitic chlorophyte. The signs of infection, together with the morphological observations, suggest that this parasitic algae may be Coccomyxa parasitica. However, further molecular studies are required for confirmation. This is the first report of Coccomyxa algae parasitizing the species M. galloprovincialis.     

Biomarker responses and accumulation of hazardous substances in mussels (Mytilus trossulus) transplanted along a pollution gradient close to an oil terminal in the Gulf of Finland (Baltic Sea)

Baltic Sea blue mussels (Mytilus trossulus) were used as sentinel organisms to detect the biological effects of chemical contamination in the low salinity environment. Mussels naturally adapted to a salinity of ca. 6.0 PSU were caged for 30 days at four sites along an assumed pollution gradient (salinity ca. 4.5 PSU) in the vicinity of Finland's largest oil refinery and harbor Kilpilahti in the Gulf of Finland. Tissue concentrations and accumulation rates of especially organic contaminants (PAHs, PCBs and organotins) were clearly elevated at the innermost coastal stations near the harbor area. Biological effects of contaminant exposure on caged mussels were evaluated by measuring a suite of biomarkers including catalase, glutathione S-transferase, superoxide dismutase, glutathione reductase, lipid peroxidation, acetylcholinesterase activity and lysosomal membrane stability. Mussels transplanted near the harbor area were able to elevate their antioxidant defense in response to environmental contamination. Reduced morphometric condition index and soft tissue growth rate together with increased lipid peroxidation and low lysosomal membrane stability were also observed at the most contaminated site. The results suggest that caging of M. trossulus for four weeks at lower salinity is a feasible method for the detection of environmental pollution also in low salinity areas of the Baltic Sea.     

Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa

The effects of cryoprotectants, cooling rate and freezing on the mussel Mytilus galloprovincialis sperm were evaluated. At the end of each step of the experimental protocol, motility and fertilization ability of sperm were analyzed, compared to fresh semen. Five cryoprotectants were tested in their toxicity level: dimethylsulfoxide, ethylene glycol, 1-2 propylene glycol at 5%, 7%, 10%, 15% and 20% concentration; glycerol and methanol at concentration of 5%, 7% and 10%. The incubation times were 10, 20 and 30 min at 20 ± 1 °C. Only dimethylsulfoxide, ethylene glycol and 1-2 propylene glycol at 5%, 7% and 10% were chosen for the following pre-freezing step. Five adaptation/chilling rates were analyzed: 10 min at 20 ± 1, −2, −1, −0.5 and −0.25 °C/min and the last one was used for testing the best freezing procedure among seven gradients. Particularly, two rapid rates, three slow rates and two double step rates were conducted.Thawing results showed that M. galloprovincialis sperm are very sensitive to rapid pre-freezing and freezing protocols and only a slow procedure assured good motility and fertilization percentages.     

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts

A battery of biochemical parameters is used to evaluate the response of mussels to a contaminated coastal environment. In the European BIOMAR programme, a multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites. AChE activity is highly sensitive to organophosphorus and carbamate insecticides but also to heavy metals. Catalase activity and lipid oxidation (evaluated as MDA) are markers of oxidative stress, GST activity is related to the conjugation of organic compounds and BPH activity is a marker of planar compounds (e.g. PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). Contamination levels were estimated by measurement of PAHs and heavy metals in animals. For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a global response index was calculated as the sum of the response index of each of the five biomarkers. As a result of our calculation method, the quality of the coastal environment at each site can be classified with a five level scale. Mussels were collected during five cruises in 1995-1996 on the Baltic and Mediterranean coasts. The results show that water quality ranged from class 1 (clean areas in some sites of the French Riviera, Spanish Costa Brava and the Baltic coast) to class 5 (high pollution in main harbours, e.g. Kiel and Toulon). Some areas fall into class 4, e.g. Carteau, Cortiou, Barcelona, Warnemunde, Swinemunde, Ebro delta. The global Biomarker Index was positively correlated with the level of PAHs in mussels in Baltic transects. A number of other contaminants or stressors may be present in the marine environment and the Biomarker Index appeared to be relevant to classify coastal environmental pollution.     

Effects of seston variability on the clearance rate and absorption efficiency of the mussels Aulacomya maoriana, Mytilus galloprovincialis and Perna canaliculus from New Zealand

The clearance rates (CR in l g⁻¹ h⁻¹) and net absorption efficiencies (AE) of three co-occurring mytilid species were estimated to determine the effects of variation in ambient seston quantity (total particulate matter, TPM, mg l⁻¹) and quality (particulate organic matter, POM, mg l⁻¹; percent organic matter, PCOM=POM/TPM) on these physiological functions. CR_S estimates were significantly different among the species (Mytilus galloprovincialis>Perna canaliculus=Aulacomya maoriana), but were not correlated with differences in species-dependent mean body size (P. canaliculus>M. galloprovincialis>A. maoriana). AE estimates were independent of mean body weight, and did not differ among the species. For all three species, CR responded in a simple linear manner to seston organic content, either in terms of POM (for A. maoriana and M. galloprovincialis), or PCOM (for P. canaliculus). The AE response, although also of a simple linear type, was species-dependent and involved interactions of two or three seston components: TPM, POM and PCOM for A. maoriana; POM and PCOM for M. galloprovincialis; and TPM and PCOM for P. canaliculus. For all three species, seston variation explained 15-20% of the variation in CR and 52-59% of the variation in AE. Comparisons among the species indicated that two very different responses to variation in seston quantity and quality exist. First, significant differences in CR result from species-dependent differences in the magnitude of the response to seston variation. Second, species-dependent responses to variation in seston variation resulted in the similarity of the AE responses. Thus, the three species appear to have evolved different strategies for dealing with seston variation, but with the end result that their AE responses do not differ. Finally, no evidence was found of a negative association between CR and AE for any of the three species, suggesting that under the seston conditions experienced in this work, these species do not have the physiological compensatory capacity to reduce CR in order to increase AE. The importance of a comparative (i.e., multi-species) approach utilising ambient seston is emphasised by the findings of this research if we are to understand better the feeding and digestive physiologies of suspension-feeding organisms such as mussels.     

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts

A battery of biochemical parameters is used to evaluate the response of mussels to a contaminated coastal environment. In the European BIOMAR programme, a multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites. AChE activity is highly sensitive to organophosphorus and carbamate insecticides but also to heavy metals. Catalase activity and lipid oxidation (evaluated as MDA) are markers of oxidative stress, GST activity is related to the conjugation of organic compounds and BPH activity is a marker of planar compounds (e.g. PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). Contamination levels were estimated by measurement of PAHs and heavy metals in animals. For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a global response index was calculated as the sum of the response index of each of the five biomarkers. As a result of our calculation method, the quality of the coastal environment at each site can be classified with a five level scale. Mussels were collected during five cruises in 1995-1996 on the Baltic and Mediterranean coasts. The results show that water quality ranged from class 1 (clean areas in some sites of the French Riviera, Spanish Costa Brava and the Baltic coast) to class 5 (high pollution in main harbours, e.g. Kiel and Toulon). Some areas fall into class 4, e.g. Carteau, Cortiou, Barcelona, Warnemunde, Swinemunde, Ebro delta. The global Biomarker Index was positively correlated with the level of PAHs in mussels in Baltic transects. A number of other contaminants or stressors may be present in the marine environment and the Biomarker Index appeared to be relevant to classify coastal environmental pollution.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Facilitation and competition between invasive and indigenous mussels over a gradient of physical stress

The interactions between invasive exotic and indigenous species can have profound harmful effects on the recipient community; however, not all such interactions are negative. Facilitation is increasingly recognised as important in shaping natural communities and is believed to vary under different conditions. Earlier studies have shown that the indigenous intertidal mussel Perna perna initially facilitates survival of the invasive Mytilus galloprovincialis in the low mussel zone by providing protection against waves, but later excludes M. galloprovincialis through interference competition for space. Here, we examined interactions between these species in the mid and upper mussel zones, moving mussels to experimental plots in different combinations of densities and species. Mussels were left on the shore for more than a year and treatment effects on mortality, shell length and condition were compared. In the high zone, treatment had no effects and P. perna showed greater mortality than M. galloprovincialis, indicating that its exclusion from the high shore is due to emersion stress. In the mid zone, treatment had no significant effects on M. galloprovincialis, but multiple comparisons among treatments involving P. perna showed that facilitation occurred. P. perna survived better at higher densities, but survived even better when mixed with the physiologically more tolerant M. galloprovincialis. Length data indicated both inter- and intraspecific competition for P. perna in the mid zone. Whereas facilitation occurs strongly in the low zone (P. perna facilitates M. galloprovincialis) and weakly in the mid zone (M. galloprovincialis facilitates P. perna), the lack of facilitation in the high zone suggests that the probability of facilitation is not linearly linked to increasing physical stress. Instead it is likely to be hump shaped: relatively unimportant under conditions that are benign for a particular species, significant under more severe conditions, and overridden by physical stress under very harsh conditions.     

The respiratory capacity of marine mussels (Mytilus galloprovincialis) in relation to the high temperature threshold

Thermal tolerance limits of ectotherms may result from respiratory limitations. In response to declining oxygen availability, organisms have shown to exhibit oxyregulation by enhancing ventilation and heartbeat rates. In this study we examined how this regulatory response in mussels (Mytilus) changes with increasing temperature. Experimental mussels showed extensive oxyregulation at temperatures near to their habitat temperature, but increasingly lost this capacity towards higher temperatures. At breakpoint temperature no regulation took place and respiration rates changed proportional to oxygen availability. These results revealed how thermal limitations relate to respiratory capacity of mussels.     

High survival and growth rates of introduced Pacific oysters may cause restrictions on habitat use by native mussels in the Wadden Sea

Pacific oysters Crassostrea gigas (Thunberg, 1793) were introduced to the northern Wadden Sea (North Sea, Germany) by aquaculture in 1986 and finally became established. Even though at first recruitment success was rare, three consecutive warm summers led to a massive increase in oyster abundances and to the overgrowth of native mussel beds (Mytilus edulis L.). These mussels constitute biogenic reefs on the sand and mud flats in this area. Survival and growth of the invading C. gigas were investigated and compared with the native mussels in order to predict the further development of the oyster population and the scope for coexistence of both species. Field experiments revealed high survival of juvenile C. gigas (approximately 70%) during the first three months after settlement. Survival during the first winter varied between > 90% during a mild and 25% during a cold winter and was independent of substrate (i.e., mussels or oysters) and tide level. Within their first year C. gigas reached a mean length of 35-53 mm, and within two years they grew to 68-82 mm, which is about twice the size native mussels would attain during that time. Growth of juvenile oysters was not affected by substrate (i.e., sand, mussels, and other oysters), barnacle epibionts and tide level, but was facilitated by fucoid algae. By contrast, growth of juvenile mussels was significantly higher on sand flats than on mussel or oyster beds and higher in the subtidal compared to intertidal locations. Cover with fucoid algae increased mussel growth but decreased their condition expressed as dry flesh weight versus shell weight. High survival and growth rates may compensate for years with low recruitment, and may therefore allow a fast population increase. This may lead to restrictions on habitat use by native mussels in the Wadden Sea.     

Nocardiosis in Mediterranean bivalves: First detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy)

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.     

Incidence of hemocytes and parasites in coastal populations of blue mussels (Mytilus edulis)--testing correlations with area,season, and distance to industrial plants

Blue mussel hemocytes (cells with immunoresponse activities) are suggested as indicators of anthropogenic contamination. We compared hemocyte numbers, granulocytoma (aggregated hemocytes), and parasites among populations of mussels from different areas of Skagerrak (a north and a south), seasons (summer and autumn), and impact levels (close or far from industrial activities). Seasonal hemocyte numbers were larger in the north compared to the south. Northern unimpacted populations had higher hemocyte numbers than populations close to industries, while no differences were found in the south. More uneven tissue distributions were found in populations far from industries in the north area and in populations close to industries in the south area. Parasites were more common in northern mussels than in southern, but no relationship to impact level was found. Mussels with granulocytoma, however, were found in all populations from the impacted sites while in none of the other populations suggesting granulocytoma as a possible indicator of industrial impact.     

Feeding activity of scallops and mussels measured simultaneously in the field: Repeated measures sampling and implications for modelling

Many studies on the biology of bivalves have focused on identifying the importance of various environmental factors in regulating feeding behaviour in order to predict ingestion and growth responses in a variety of habitats. When taking into consideration the diversity of studies undertaken and the variety of methodologies employed, it is perhaps not surprising that there have been inconsistencies in both interspecific and intraspecific comparisons. In order to compare and contrast the way in which feeding activities of a mussel species (Mytilus edulis) and a scallop species (Placopecten magellanicus) respond to environmental factors and seston characteristics, we exposed both species simultaneously to a fluctuating assemblage of natural particles found in their local environment. Similar sizes of mussels (40-73 mm) and scallops (40-88 mm) were held in flow through chambers at two different sites in Atlantic Canada on four separate occasions, during which environmental conditions, including seston characteristics, were monitored and individual clearance rates were measured. Because the same individuals were measured over time, repeated-measures statistical procedures were used to analyze the data. Results indicate that weight standardized clearance rates of mussels were significantly higher than those of scallops only at the highest concentration tested (12.8 mg l^− 1), which translates into almost 3 times more material being filtered. Scallops appeared to respond to fluctuations in environment parameters and suspended food particles more consistently than mussels. These data suggest that the feeding behaviour of these two species is mediated differently by the same environmental parameters. Our study also demonstrates the importance of selecting appropriate statistical tests (e.g., repeated measures) for the analyses of data obtained from repeated sampling of the same individuals over time, and the need to consider species-specific feeding responses when developing models that predict the influence of bivalve feeding on ecosystem function.     

Nucleic acid indices of egg production in the tropical copepod Acartia sinjiensis

Two experiments were conducted to evaluate the effects of food and temperature on the nucleic acid content and egg production (and their relationship) of the tropical copepod Acartia sinjiensis. Experiment 1 evaluated the effect of food quality (as different algae species) on the relationship between nucleic acid content and egg production. In Experiment 2, the main and interaction effects of food type, food concentration and temperature on the total, Carbon and Nitrogen specific egg production and nucleic acid indices were evaluated in a factorial experimental design. Food quality, concentration and temperature significantly affected the nucleic acid content, egg production rate and the nucleic acid-egg production relationship of A. sinjiensis. RNA indices were correlated with egg production in females fed Pavlova salina, Tetraselmis chuii and Chaetoceros muelleri, but not in females fed Isochrysis aff. galbana. The slopes of the linear regressions of RNA indices as predictors of egg production were similar in females fed different algae species, suggesting that the slope of these relationships might be independent of food quality. The DNA content of females was significantly affected by food and temperature, suggesting that it is not a good index of cell number in this species. Nevertheless, the RNA:DNA ratio was as good a predictor of egg production as total RNA content. Egg production showed a weakly positive correlation with temperature. On the other hand, total, C- and N-specific nucleic acid indices had a strong negative correlation with temperature. In addition, temperature had a non linear effect on the slopes of the regression lines of RNA content and RNA:DNA ratio as predictors of egg production—slopes were similar at 25 °C and 30 °C, but significantly lower at 20 °C. Furthermore, the predictability of egg production was improved when the interaction term of nucleic acid indices with temperature was used instead of the nucleic acid indices alone in linear regression models. Our results suggest food quality has a limited influence on the nucleic acid-egg production relationship, and that temperature should be accounted for in models using nucleic acid indices as predictors of egg production in A. sinjiensis.     

Effects of experimental harvesting on recruitment of an alien mussel Mytilus galloprovincialis

Successful fisheries management is underpinned by an understanding of the processes that underlie the population dynamics of exploited stocks. This study considered the effects of experimental harvesting on recruitment of Mytilus galloprovincialis along the west coast of South Africa, where harvesting of this alien species is being contemplated. In particular, the role of settlement habitat availability in the form of adult mussels was analysed. To track the effects of a spectrum of harvesting intensities, five treatments were implemented: F = 0 (i.e. a control), F = 0.3, F = 0.6, F = 0.9 and F = 1. At these harvesting intensities 0%, 30%, 60%, 90% or 100% of mussel biomass was removed respectively at the outset of the experiment. A significant negative exponential relationship (p < 0.01) was found between M. galloprovincialis recruit density and harvesting intensity, with intensities greater than F = 0.3 dramatically reducing recruitment. This pattern was recorded throughout the intertidal zone and remained temporally constant over 2 years. Significant positive linear relationships (p < 0.01) between recruit density and adult mussel biomass or density indicate a strong correlation between availability of settlement habitat and recruitment. It is likely that the high recruit density recorded at low harvesting intensities (2000-20 000 per 0.01 m²) exceeds the level required for population maintenance. However, if settlement habitat is eliminated or significantly reduced, as was achieved by F = 0.3 or above, recruitment may become limiting. Thus, to protect stock replenishment, harvesting of M. galloprovincialis in this region should take place at intensities less than F = 0.3.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.