Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus > V. anguillarum > M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+ 35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+ 16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus > V. anguillarum > M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.     

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.     

Mass mortality in a population of the mussel Mytilus edulis L. Caused by high temperature on rocky shores

Mass mortality in a population of the mussel Mytilus edulis L. occurred in early August, 1981, on rocky intertidal shores in Mutsu Bay, northern Japan, due to unusually high temperatures. The decomposed tissues disappeared within 3 days after death. Higher mortality was observed in the upper part of the Mytilus zone than in the lower zone. As temperatures rose the mussels expelled water contained in the shells and 50% individuals died within 1 h from high body temperature rising up to >40°C. Septifer (Mytilisepta) virgatus (Wiegmann), which occupied the zone above Mytilus, showed low mortality (maximum 16.8%). Heat tolerance seems stronger in Septifer than in Mytilus. Many small animals including polychaetes, amphipods and nemerteans etc., lived among the empty shells and when the shells were removed by wave action, these animals disappeared from the bared rock surface. One year after the mass mortality the rock surface was covered by the barnacle Chthamalus challengeri Hoek. The ecological effect of mass mortality on the intertidal community is discussed.     

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.     

The effects of feeding Karlodinium veneficum (PLY # 103; Gymnodinium veneficum Ballantine) to the blue mussel Mytilus edulis

The effects of exposure to the type species for Karlodinium veneficum (PLY # 103) on immune function and histopathology in the blue mussel Mytilus edulis were investigated. Mussels from Whitsand Bay, Cornwall (UK) were exposed to K. veneficum (PLY # 103) for 3 and 6 days. Assays for immune function included total and differential cells counts, phagocytosis and release of extra cellular reactive oxygen species. Histology was carried out on digestive gland and mantle tissues. The toxin cell quota for K. veneficum (PLY # 103) was measured by liquid chromatography–mass spectrometry detecting two separable toxins KvTx1 (11.6 ± 5.4 ng/ml) and KvTx2 (47.7 ± 4.2 ng/ml). There were significant effects of K. veneficum exposure with increasing phagocytosis and release of reactive oxygen species following 6 days exposure. There were no significant effects on total cell counts. However, differential cell counts did show significant effects after 3 days exposure to the toxic alga. All mussels produced faeces but not pseudofaeces indicating that algae were not rejected prior to ingestion. Digestive glands showed ingestion of the algae and hemocyte infiltration after 3 days of exposure, whereas mantle tissue did not show differences between treatments. As the effects of K. veneficum were not observed in the mantle tissue it can be hypothesized that the algal concentration was not high enough, or exposure long enough, to affect all the tissues. Despite being in culture for more than 50 years the original K. veneficum isolate obtained by Mary Parke still showed toxic effects on mussels.     

Yessotoxin detected in mussel (Mytilus californicus) and phytoplankton samples from the U.S. west coast

Yessotoxin (YTX) was detected in an algal sample and two mussel samples (0.07–0.10 μg g⁻¹) collected from Scripps Pier in La Jolla, California during a bloom of Lingulodinium polyedrum. Mussel samples collected from Monterey Bay, California also contained measurable YTX (levels up to 0.06 μg g⁻¹) in samples obtained during a 6-month (weekly) sampling period. Gonyaulax spinifera and L. polyedrum were identified in background concentrations in Monterey Bay during the time of contamination. An algal sample from Washington coastal waters collected during non-bloom conditions also contained YTX, possibly originating from Protoceratium reticulatum.Three strains of L. polyedrum (CCMP1931, CCMP1936, 104A) isolated from southern California coastal waters and one strain of G. spinifera (CCMP409) isolated from Maine were tested for YTX production using two methods, competitive ELISA and liquid chromatography–mass spectrometry (LC–MS). The ELISA method detected YTX in the particulate phase in two of three L. polyedrum strains. The LC–MS method did not detect YTX in the particulate or dissolved phase of any of the strains.To our knowledge, this is the first study to test and confirm YTX in environmental samples from California and Washington coastal waters. It is highly likely that L. polyedrum was responsible for the YTX contamination in the southern California samples. Future research needs to conclusively determine the biological origin(s) of YTX contamination in central California and Washington coastal waters.     

Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na/H exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides (O₂⁻) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na⁺/H⁺ exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O₂⁻ and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O₂⁻ and NO generation in haemocytes of mussels. Moreover, O₂⁻ and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O₂⁻ production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O₂⁻ or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O₂⁻ and NO production. The involvement of NHE in the induction of O₂⁻ and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.     

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).     

Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.     

The establishment of fucoid zonation on algal-dominated rocky shores: hypotheses derived from a simulation model

1. A model was developed for the growth of intertidal algae with photosynthesis simulated both in air and in a tidal water column. Morphological data on dry mass per unit area and length–area relationships were used to separate the growth of different fucoid species. The relative growth rate of fronds at any height on the shore depended on a trade-off between net photosynthetic performance and tolerance to desiccation.
2. The simulated zonation patterns and growth rates were consistent with those observed previously for Fucus spp. and Pelvetia canaliculata .
3. The simulated growth of Ascophyllum nodosum was always slower than for the other species. This species did not form its characteristic distribution zones in simulations without including further processes in the model. However, Ascophyllum collected from the field could be separated into upper and lower shore morphologies which formed separate zones when they were simulated in competition with each other.
4. Several hypotheses were proposed concerning the relative locations and sharpness of interspecies boundaries on the shore. Zonation patterns were relatively insensitive to changes in most model parameters except the desiccation rate.     

Invasion of rocky shores by a mytilid mussel reveals an abundant-centre distribution coupled with moderate increases in densities at its absolute range limits

Semimytilus patagonicus is an invasive mussel on the coast of southern Africa and has extended its range in recent years. We asked whether its distribution and abundance are consistent with the abundant-centre hypothesis (ACH). Marginal populations were located by monitoring 33 rocky shore sites in South Africa and southern Namibia in 2021. This revealed no changes to its distributional limits since 2020. At nine of these sites, population demography was measured to allow a comparison of their densities and size structure. Four were central populations on the west coast of South Africa (including the site where the species was first detected in 2009). Four were marginal populations in South Africa: two towards the cold range edge in the north and two towards the warm range edge to the south. The ninth population was in southern Namibia, representing a recent invasion event first detected in 2014. Across the species' South African range, the distribution of its abundance was generally consistent with the ACH, with the greatest abundance at its range centre and a gradual decrease towards the range edges. However, the ultimate marginal population at both its cold and warm range edges showed moderate upticks in abundance compared to the penultimate marginal populations. Additionally, marginal populations in South Africa typically included a greater proportion of large individuals. Recruitment intensity was greater in warm range edge populations than cold range edge populations. The size structure of the population in Namibia resembled those of central populations in South Africa. Moderate increases in densities at the absolute range limits suggest that the species is currently undergoing spread into regions associated with moderately optimal environmental conditions (ultimate range edge sites) after encountering regions associated with suboptimal environmental conditions (penultimate range edge sites).     

Molecular cloning of the ribosomal P-proteins MgP1, MgP2, MgP0, and superoxide dismutase (SOD) in the mussel Mytilus galloprovincialis and analysis of MgP0 at stress conditions

The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.     

Resilience and elasticity of intertidal communities in response to harvesting of the alien mussel Mytilus galloprovincialis

Besides the direct impacts of exploitation on target species, indirect effects on non-target species are unavoidable and find expression in changes in community structure. We quantified the effects of experimental harvesting of Mytilus galloprovincialis on intertidal communities on the South African west coast. In the mid- and low-shore, four months of harvesting at intensities greater than F = 0.3 and F = 0.6 respectively, resulted in significant changes in community composition. These changes were driven by progressively greater spatial dominance by the macroalgae Cladophora flagelliformis, Porphyra capensis and Ulva species as harvesting intensity increased. Four months after cessation of harvesting, community structure had not recovered and even areas subjected to as little as F = 0.3 supported significantly altered communities in both zones. The fact that substantial community changes were induced by even low-intensity exploitation is indicative of low resilience to harvesting. The densities and cover of the dominant taxa returned to a pre-harvest state 16 months after the cessation of harvesting, but multivariate analyses indicated that the overall community composition required 32 months for Cover Letter full recovery. Although these communities displayed elasticity within three years, it is recommended that should a M. galloprovincialis fishery be established in the region, harvesting be implemented at a maximum intensity of F = 0.3. This approach would maximise yields and protect associated intertidal communities.     

The effects of acetylcholine, dopamine and 5-hydroxytryptamine on water pumping rate and pressure in the mussel Mytilus edulis L

Acetylcholine (ACh) and dopamine (DA), at certain concentrations, are known inhibitors of ctenidial lateral cilia. Addition of either reduces water pumping rate in intact Mytilus edulis by a maximum of 60–70%. The remaining 30–40% of flow is probably generated by the abfrontal cilia which are unaffected by either substance. Reduced pumping rate results in a reduction of exhalant aperture size which almost maintains exhalant water velocity. Exhalant pressure is slightly reduced after addition of either substance. Addition of 5-hydroxytryptamine (5-HT) causes an initial small reduction in water pumping rate, but rate recovers to pre-addition levels after 10–15 min.     

Phytoplankton composition of the Kandalaksha Gulf, Russian White Sea: Dinophysis and lipophilic toxins in the blue mussel (Mytilus edulis)

Dinophysis acuminata and D. norvegica were observed in plankton net samples during the summer of 2002 from the Kandalaksha Gulf in the White Sea (North European Russia). Prorocentrum lima was found as an epiphyte on subtidal macroalgae in August, but not observed in plankton net samples. Protein phosphatase 2A (PP2A) inhibition measured 127.8 ng OA-equivalent/g of mussel (Mytilus edulis) hepatopancreas from samples collected a few days after when Dinophysis was recorded at a density of 1550 cells L⁻¹. Liquid chromatography–mass spectrometry confirmed presence of several classes of lipophilic shellfish toxins associated with Dinophysis spp. in the mussels including okadaic acid, dinophysistoxin-1, pectenotoxins and yessotoxins. No azaspiracid was detected. This represents the first identification of phycotoxicity in the White Sea.     

“Survival in air” of the blue mussel Mytilus edulis L. as a sensitive response to pollution-induced environmental stress

Mussels, Mytilus edulis, were exposed for periods of 6 weeks at various locations in Dutch coastal waters during 1989 and 1990. “Survival in air” showed to be a sensitive response parameter for indicating pollution induced environmental stress in transplanted mussels sampled from eight field sites. Increased tissue contaminant levels, especially PCBs and PAHs, correlated with a reduced survival time during aerial exposure. Three weeks exposure of mussels in the laboratory to 1 μg · 1⁻¹ PCBs affected the aerial survival time negatively. Laboratory experiments did not indicate that lowered salinity influences the “Survival in air” response after sufficient acclimation (15 days), facilitating the use of this response parameter in both marine and estuarine waters.     

Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: Histopathology, immune responses, and recovery

Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods.Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense.No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.     

Spatial patterns of growth in the mussel, Mytilus californianus, across a major oceanographic and biogeographic boundary at Point Conception, California, USA

The Point Conception, California, USA region (hereafter PC) is one of the most important biogeographic and oceanographic discontinuities on the US west coast. Here we address how mesoscale oceanographic variability in the region around PC affects the growth of the competitively dominant species in the rocky intertidal assemblage: the mussel Mytilus californianus. Strong upwelling and high wave exposure dominate the California coast north of PC, and weak, seasonal upwelling and warmer water temperatures are characteristic of the region south/east of PC. We hypothesized that the oceanographic gradients in temperature, upwelling, wave exposure and productivity around PC would exert strong bottom–up influences on growth rates of mussels, potentially underlying large-scale differences in community structure around the PC region. We evaluated these predictions by measuring mussel growth rates across the PC region both in the intertidal and offshore on moorings. Intertidal mussels grew at much higher rates at sites south relative to north of PC and growth rates decreased in a gradient from south to north. The gradient in intertidal mussel growth around PC was uncorrelated with inshore concentrations of chlorophyll-a, and was most strongly correlated with the alongshore gradient in wave exposure and intertidal temperature. Mussels on moorings offshore from the intertidal sites grew at much higher rates than those in the corresponding intertidal areas, and mussel growth rates did not differ significantly among moored locations around PC. The gradient of increasing temperature from north to south among mooring sites was correlated with a decreasing gradient in productivity in the same direction, potentially contributing to equal and opposite effects on mussel growth at offshore moorings. This study suggests that environmental factors such as cold temperatures and high wave exposure contribute to the spatial pattern of decreasing mussel growth rates from south to north around PC, underlying large-scale patterns of community structure in this region.