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.     

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.     

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.     

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).     

Stable isotope fractionation in the digestive gland, muscle and gills tissues of the marine mussel Mytilus galloprovincialis

Mytilus galloprovincialis is a common species in the Mediterranean. It is a sedentary filter-feeding organism that assimilates carbon and nitrogen isotopic ratios in tissues from its food sources. The δ¹³C and δ¹⁵N values have been used to demonstrate differences in isotopic composition between digestive gland, muscle and gills of this mussel. For δ¹³C, mean values were - 21.99 ± 0.50‰, - 19.70 ± 0.44‰, and - 19.96 ± 0.44‰, respectively; and for δ¹⁵N, they were 5.16 ± 0.90‰, 7.67 ± 0.79‰ and 7.77 ± 0.85‰, respectively. The fractionation or enrichment factor for ¹³C values between digestive gland and muscle, between digestive gland and gills, and between muscle and gills were - 2.29 ± 0.16‰, - 2.04 ± 0.14‰ and 0.27 ± 0.07‰, respectively, within the expected range of ¹³C fractionation at filter feeders reported elsewhere. In contrast, low fractionation values were found for ¹⁵N with - 2.45 ± 0.24‰, - 2.51 ± 0.16‰ and - 0.11 ± 0.16‰, between digestive gland and muscle, between digestive gland and gills, and between muscle and gills, respectively. Through isotopic fractionation of M. galloprovincialis, the depleted values were found in the digestive gland, followed by gills and then by muscle tissue. Statistical analysis (PERMANOVA) was performed to check for significant differences in δ¹³C and δ¹⁵N isotopic signatures between tissues and localities. The current study demonstrates significant differences in the δ¹³C and δ¹⁵N isotopic composition between digestive gland, muscle and gills tissues in M. galloprovincialis living in the oligotrophic environment of the Balearic Islands.     

Competition and facilitation between the alien mussel Mytilus galloprovincialis and indigenous species: Moderation by wave action

As the alien species that most dominates space along the South African coast, the Mediterranean mussel Mytilus galloprovincialis has radically altered community composition on invaded shores. We experimentally assessed interspecific interactions between this invasive species and dominant indigenous species in conjunction with considering how wave action moderates such interactions. The density of both M. galloprovincialis and the limpet Scutellastra granularis increased with wave action. Conversely, the tube-building polycheate Gunnarea capensis was negatively affected by wave exposure, being most abundant on sheltered shores. The influence of wave action on the indigenous mussel Aulacomya ater, however, remains unclear. M. galloprovincialis outcompeted both G. capensis and A. ater at moderate to high exposure levels, whereas it had both positive and negative effects on S. granularis. It outcompeted adult limpets on primary rock space on semi-exposed and exposed shores, reducing densities of this portion of the population. However, recruitment of S. granularis was facilitated by M. galloprovincialis, as greater numbers recruited to the secondary substratum offered by mussel shells. Again this interaction intensified with wave action. Due to the extremely high density of recruits on secondary space, the net effect of M. galloprovincialis on S. granularis was positive. Thus, wave action not only influences the abundance of individual species, but also mediates both positive and negative interspecific interactions in rocky shore communities, including the impact of alien species such as M. galloprovincialis.     

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.     

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.     

Carbonic anhydrase activity in Mytilus galloprovincialis digestive gland: Sensitivity to heavy metal exposure

Heavy metals are known to in vitro inhibit carbonic anhydrase (CA) activity in a variety of organisms; however, little is known about their in vivo effects on the activity and the expression of this metalloenzyme. The aim of this work was to investigate the in vitro and in vivo sensitivity to cadmium of CA in the digestive gland of Mytilus galloprovincialis. CA activity and protein expression (apparent molecular mass of about 28 kDa) were demonstrated in mussel digestive gland for the first time. CA activity showed week sensitivity to in vitro cadmium exposure, while it was significantly increased (about 40%) following two weeks in vivo exposure. In parallel, CA protein expression appeared significantly enhanced as demonstrated by Western blotting. Laboratory experimental results were confirmed by a field experiment. Mussels exposed for 30 days to an impacted site showed a significant increase of the CA activity and protein expression with respect to animals exposed to the control site in parallel to the increase of the metallothionein tissutal concentration. In conclusion in the present work for the first time CA activity and protein expression have been demonstrated to be enhanced by the exposure to the trace element cadmium in animals.     

Changes in fatty acid composition of Mytilus galloprovincialis (Lmk) fed on microalgal and wheat germ diets

Dietary fatty acid incorporation and changes in various lipid and phospholipid classes in the mussel Mytilus galloprovincialis subjected to three different dietary regimens were analysed and compared. Group A was unfed; group B received a diet consisting of 100% Thalassiosira weissflogii, exhibiting the typical fatty acid composition of diatoms, and group C received a diet consisting of 100% wheat germ conferring a 18:2:n-6 abundance. Biochemical analyses of diets and mussels were carried out at the beginning and at the end of the 30-day experimental period. Starvation and T. weissflogii based diet poorly affected mussel growth and fatty acid composition which remained unchanged. On the contrary, the wheat germ-based diet increased the condition index and deeply affected the fatty acid profile of all lipid and phospholipid classes. The high dietary 18:2n-6 level drastically reduced tissue content of 20:4n-6, 20:5n-3 and 22:6n-3. The biosynthesis of Non Methylene Interrupted (NMI) dienoic fatty acid appeared to be insensitive to the high input of 16:1n-7 and 18:1n-9 respectively from diet B and C, and to the PUFA shortage of diet C. Nevertheless the two NMI trienoic derivatives, 20:3Δ5,11,14 and 22:3Δ7,13 16, were found higher in C with respect to other groups, presumably due to the high 18:2n-6 content of this diet.     

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.     

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.     

Prey productivity effects on the impact of predators of the mussel, Mytilus californianus (Conrad)

The relationship of increasing prey productivity, a measure incorporating prey settlement and body growth, to changes in the relative impact of two predator groups, birds and the sea star, Pisaster ochraceus (Brandt) on a competitively dominant mussel, Mytilus californianus were examined. The purpose of this experiment was twofold, 1) to determine if the separate effects of each predator group on prey abundance increased as prey productivity increased and 2) to determine if the relative impacts of the two predator groups diverged as prey productivity increased. In this experiment, the separate impact of each predator group increased with increasing prey productivity. However, the relative impact of each predator group did not diverge with increasing prey productivity. Unlike previous studies that suggested with increasing prey productivity the relative effect of two predator groups should diverge, this experiment suggested that communities can have more redundant predator groups than originally thought. The results of an analysis using a proportional hazards model suggested that despite increasing prey productivity, birds and the sea star were equal in their ability to curb population increases by M. californianus. These results highlight the need to carefully consider what type of species to species comparisons to make when attempting to discern the relative roles of different predator groups in a community.     

Larval settlement can explain the adult distribution of Mytilus californianus Conrad but not of M. galloprovincialis Lamarck or M. trossulus Gould in Moss Landing, central California: Evidence from genetic identification of spat

We investigated the spatial distribution of adult and newly settled mussels (Mytilus galloprovincialis Lamarck, Mytilus trossulus Gould and Mytilus californianus Conrad) on the shore at Moss Landing, California to test the hypothesis that adult distributions are a result of settlement patterns. Adult M. californianus were most abundant on a wave-exposed rocky jetty and adults of Blue mussels (M. trossulus and M. galloprovincialis) were more abundant inside the protected Moss Landing harbor. Using taxon-specific polymerase chain reactions, we monitored recruitment during continuous 1-2 week intervals on fibrous scrubbing pads for 12 months in 2002-2003. All mussel species settled in greatest numbers on the exposed jetty, and Blue mussels settled in greater numbers there than did M. californianus. Because Blue mussels settled abundantly where their adults were rare, post-settlement mortality appeared to be the strongest influence on adult distribution. In contrast, M. californianus settled mostly in their adult habitat.     

Assessing the potential benthic-pelagic coupling in episodic blue mussel (Mytilus edulis) settlement events within eelgrass (Zostera marina) communities

Coastal marine seagrass ecosystems are important nursery grounds for commercially and recreationally important species, and they serve as key settlement and recruitment sites for other species. We investigated several years (2001-2003) where episodic settlement events of blue mussels (Mytilus edulis) occurred in Barnegat Bay, NJ, USA. Population assessment indicated that blue mussels settled in eelgrass beds (Zostera marina) in late spring with peak densities exceeding 170,000 m⁻². Based on calculated filtration rates of M. edulis, we determined that for at least 53 days in 2001, the density and size distribution of M. edulis were sufficient to filter the water column volume in excess of twice a day, with maximum calculated filtration rates exceeding 8 m³ water m⁻² day⁻¹. While the settlement event in 2001 was very localized, in 2003, the settlement event was considerably more widespread throughout the bay, with maximum settling densities exceeding 175,000 individuals m⁻². Associated with these high densities, maximum calculated filtration rates exceeded 15 m³ water m⁻² day⁻¹. This filtration potential may have impeded the localized development of a brown-tide (Aureococcus anophagefferens) bloom in 2001, which occurred in other regions of the bay, but the widespread settlement event seen in 2003 may have impeded the development of any brown-tide blooms in Barnegat Bay during that summer. The decline in mussel densities throughout the summer may be a result of elevated water temperatures in this back bay, but at one site, the high settlement of M. edulis was followed by a substantial migration (>40 individuals m⁻²) of small sea stars (Asterias forbesii). In 2001, A. forbesii was a significant factor in reducing M. edulis density by the end of the summer at the Barnegat Inlet site and a community level assessment showed significant positive correlations between mussel aggregations and sea star densities (r=0.68-0.73, P<0.001). At this same site in 2003, the sea stars were again present in high densities (26 m⁻²) and were a potential mechanism for mussel decline. In other regions of the bay, sea star densities are very low, but numerous other predatory species exist, including blue crabs (Callinectes sapidus), green crabs (Carcinus maenus), spider crabs (Libinia spp.), and several Xanthid crabs. Given the high mussel densities seen in this study and the considerable predation by sea stars and other benthic predators, the benthic-pelagic coupling which these mussels provide in this system contributes to the high secondary production in these grass beds.     

Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels (Mytilus galloprovincialis) were experimentally contaminated with paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense, and changes in toxin content and specific composition during the decontamination period were analyzed by high-performance liquid chromatography (HPLC). Toxins excreted by the mussels into the seawater were also recovered using an activated charcoal column and analyzed by HPLC. The predominant toxins in A. tamarense, mussels, and seawater were the N-sulfocarbamoyl-11-hydrosulfate toxins (C1,2) and carbamate gonyautoxins-1,4 (GTX1,4). There were no remarkable differences in the relative proportions of the predominant toxins within A. tamarense, mussels and seawater. Because the relative proportion of the various toxin analogues excreted by the mussels was similar to that within their tissues during detoxification, it appeared that the selective release of particular toxins by the mussels was unlikely. The total amount of toxin lost from mussels was nearly equal to that which was found dissolved in the seawater, suggesting that, at least the early stages of mussel detoxification, most losses can be accounted for by excretion.     

A comparative study of the fatty acid profile of Artemia franciscana and A. persimilis cultured at mesocosm scale

The goal of this study was to examine the fatty acid (FA) profile of two Artemia species, A. persimilis (Argentina) and A. franciscana (Great Salt Lake,Utah; USA) in coexistence at mesocosm scale. The experiment was carried out to 1) evaluate putative differences in the fatty acid composition of both species while they share resources and 2) to investigate the causes of such differences. Although the coexistence of these species in nature has not yet been observed, it remains possible that this situation may arise in the future mainly due to the invasive ability of A. franciscana. FA analyses were performed on individuals as well as on pooled biomasses of each species, and integrated in multivariate principal components analysis (PCA). Comparison of the relative abundance of FA between the two species revealed that interspecific differences in FA composition are greater than intraspecific variability. Higher percentages of unsaturation were found in the fatty acids of A. persimilis compared to A. franciscana, demonstrating that aside from a high phenotypic effect of diet on the FA composition of the animals, a species-specific genotypic effect should not be discarded.     

Larval recruitment of the blue mussel Mytilus edulis: The effect of flow and algae

The mussel Mytilus edulis settlement and distribution was studied on plastic panels with manipulated flow regime (faired, bluff, split and angled) with or without water soluble metabolites of the green alga Cladophora rupestris. The panels were exposed vertically on a device (hydrovane) that ensures their constant orientation in the current during the peak of larval settlement at 1 m depth. In order to investigate larval distribution on the panels, half of them were coated with a silicone vacuum grease that prevents larvae from de-attachment. This grease was not toxic and did not attract or repel larvae. Low densities of larvae on the un-greased plates compared to the greased ones suggested that some of larvae left the substratum. The blue mussel larvae initially settled in regions of reduced shear velocity and then redistribute to the regions of high shear velocity. The presence of the alga increased the density of blue mussel larvae and changed their distribution on the panels. Overall, our results demonstrated that larval recruitment of M. edulis is an active process affected both by boundary-layer hydrodynamics and algal waterborne compounds.