Proteomics Identification of Azaspiracid Toxin Biomarkers in Blue Mussels, Mytilus edulis

Azaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Molecular & Cellular Proteomics 8: 1811-1822, 2009.

Reproductive Patterns and Tidal Effects on Spat Settlement of Mytilus Edulis Populations in Dundrum Bay, Northern Ireland

The gametogenic and spat settlement patterns of two Mytihis edulis beds were studied in Dundrum Inner Bay, Northern Ireland. There was evidence of gonad development throughout the year with the main development period between November and March. Spawning was protracted, lasting from May to November. Slight inter-annual and inter-population differences in the riming of the phases were observed but the cycles at both beds were broadly similar to each other and to those of other British and Irish sites. Settlement occurred throughout the year and there was evidence of both primary and secondary spat settlement at both sites. Although the reproductive cycles were similar, distinct seasonal and inter-site differences in spatfall were apparent. At the Downshire Bridge bed, settlement peaked during summer and was dominated by spat in the 0•;5-1•0 mm size range. At Ballykinler, settlement levels were highest in the winter months and larger (>1 mm) spat dominated the samples. The orientation of spat collection pads also significantly affected numbers of the larger (>1 mm) spat. Collectors facing the flood tide attracted significantly more secondary settlers than ebb-facing collectors. This effect varied seasonally and was greater at the Ballykinler bed. It is suggested that hydrodynamic regimes may be an important factor in the differences in settlement patterns of M. edulis.

The assimilation and chronic effects of sub-lethal concentrations of Endosulfan on condition and spawning in the common mussel Mytilus edulis

The rate of uptake of Endosulfan by Mytilus edulis L. exposed to pesticide concentrations of 0.1, 0.5, and 1.0 mg/l, and its subsequent elution on removal to clean sea water, was investigated. Higher residue levels were recorded for mussels exposed to higher concentrations of the pesticide, but concentration factors were reduced. There was a rapid initial fall in tissue residue levels on transfer to clean sea water due, it is suggested, to elution of Endosulfan adsorbed on particulate matter assimilated in the gut. The spawning period was prolonged at higher concentrations and, at 1.0 mg/l, the onset of spawning was delayed, possibly due to interference with gamonic action. At 0.1 mg/l, the minor protraction of the spawning period may reflect the effect of experimental tank conditions. No seasonal trend was obvious, and there was an exaggeration of the expected fall in condition in mussels exposed to higher concentrations of Endosulfan. In controls, the expected seasonal trend was reduced.

The effect of pesticides on Byssus formation in the common mussel, Mytilus edulis

Seed mussels (Mytilus edulis) were exposed to a range of pesticides and PCBs, several of which caused a reduction in byssal attachment at higher concentrations. In queen scallops (Chlamys opercularis) byssus formation was similarly affected although this species was more sensitive than M. edulis. The sensitivity of mussels was greater at higher temperatures and decreased with increase in size. Of the compounds tested Endosulfan (organochlorine) was the most toxic, causing a 50% reduction in byssal attachment after 24 h at 0•45 mg/l. Trichlorphon (organophosphate) was the least toxic and did not affect byssal attachment at concentrations up to 30 mg/l. The probable cause of decreased byssal attachment is a reduction in pedal activity, although it is possible that direct interference with the synthesis or combination of byssus components may be involved. It is suggested that byssogenesis tests offer a rapid and convenient technique for the routine screening of potential marine pollutants.

Voltage-activated currents in cardiac myocytes of the blue mussel, Mytilus edulis

Voltage-sensitive ionic currents were identified and characterised in ventricular myocytes of the bivalve mollusc, Mytilus edulis, using the whole-cell patch-clamp technique. Two outward currents could be distinguished. A potassium A current (I-A) activated at - 30 mV from a holding potential of - 60 mV. This transient current was inactivated by holding the cells at a potential of - 40 mV and was also blocked by applying 4-aminopyridine (3 mM) to the external bath solution. A second current was identified as a delayed rectifier (I-K). This also activated at - 30 mV but exhibited a sustained time course and was still activated at a holding potential of - 40 mV. Both outward currents were reduced in the presence of tetraethylammonium ions (30 mM). A small number of heart cells also showed an inward sodium current (I-Na). This current appeared at potentials more positive than - 50 mV, reached a maximum at - 20 mV, and decreased with further depolarisation. I-Na was inactivated at a holding potential of - 40 mV and was blocked by tetrodotoxin (1 mu M). A second inward current had a sustained time course and was not inactivated by holding the cell at a potential of -40 mV, and was also not abolished by tetrodotoxin. This current peaked at 0 mV, decreasing with further depolarisation. Furthermore, it was enhanced by the addition of barium ions (3 mM) to the bath and was blocked by external cobalt (2 mM) or nifedipine (15 mu M) These findings are consistent with this being an L-type calcium current (I-Ca) The possible physiological roles of these currents in M. edulis heart are discussed. (C) 1999 Elsevier Science Inc. All rights reserved.

Simultaneous, long-term monitoring of valve and cardiac activity in the blue mussel Mytilus edulis exposed to copper

Valve and cardiac activity were simultaneously measured in the blue mussel (Mytilus edulis) in response to 10 d copper exposure. Valve movements, heart rates and heart-rate variability were obtained non-invasively using a Musselmonitor(R) (valve activity) and a modified version of the Computer-Aided Physiological Monitoring system (CAPMON; cardiac activity). After 2 d exposure of mussels (4 individuals per treatment group) to a range of dissolved copper concentrations (0 to 12.5 mu M as CuCl2) median valve positions (% open) and median heart rates (beats per minute) declined as a function of copper concentration. Heart-rate variability (coefficient of variation for interpulse durations) rose in a concentration-dependent manner. The 48 h EC50 values (concentrations of copper causing 50% change) for valve positions, heart rates and heart-rate variability were 2.1, 0.8, and 0.06 mu M, respectively. Valve activity was weakly correlated with both heart rate (r = 0.48 +/- 0.02) and heart-rate variability (r = 0.32 +/- 0.06) for control individuals (0 mu M Cu2+). This resulted from a number of short enclosure events that did not coincide with a change in cardiac activity. Exposure of mussels to increasing copper concentrations (greater than or equal to 0.8 mu M) progressively reduced the correlation between valve activity and heart rates (r = 0 for individuals dosed with greater than or equal to 6.3 mu M Cu2+), while correlations between valve activity and heart-rate variability were unaffected. The poor correlations resulted from periods of valve flapping that were not mimicked by similar fluctuations in heart rate or heart-rate variability. The data suggest that the copper-induced bradycardia observed in mussels is not a consequence of prolonged valve closure.

The initial mode of action of copper on the cardiac physiology of the blue mussel, Mytilus edulis

Previous studies have shown that low levels of copper (down to 0.8 muM) induce bradycardia in the blue mussel (Mytilus edulis) and that this is not caused by prolonged Valve closure. The aim of this study was to determine the precise mechanism responsible. To establish if copper was directly affecting heart cell physiology, recordings of contractions from isolated ventricular strips were made using an isometric force transducer, in response to copper concentrations (as CuCl2) ranging between 1 muM and 1 mM. Inhibition of mechanical activity only occurred at 1 mM copper, suggesting that the copper-induced bradycardia observed in whole animals cannot be attributed to direct cardiotoxicity. Effects of copper on the cardiac nerves were subsequently examined. Following removal of visceral ganglia (from where the cardiac nerves originate), exposure to 12.5 muM copper had no effect on the heart rate of whole animals. The effect of copper on the heart rate of mussels could not be abolished by depletion of the monoamine content of the animal using reserpine. However, pre-treatment of the animals with alpha -bungarotoxin considerably reduced the sensitivity of the heart to copper. These results indicated that the influence of copper on the heart of M. edulis might be mediated by a change in the activity of cholinergic nerves to heart. In the final experiments, mussels were injected with either benzoquinonium or D-tubocurarine, prior to copper exposure, in an attempt to selectively block the inhibitory or excitatory cholinoreceptors of the heart. Only benzoquinonium decreased the susceptibility of the heart to copper, suggesting that copper affects the cardiac activity of blue mussels by stimulating inhibitory cholinergic nerves to the heart. (C) 2001 Elsevier Science B.V. All rights reserved.

Determining optimal duration of seed translocation periods for benthic mussel (Mytilus edulis) cultivation using physiological and behavioural measures of stress

During the benthic cultivation process of Mytilus edulis (blue mussels), wild mussel seed is often transplanted from naturally occurring subtidal beds to sheltered in-shore waters to be grown to a commercial size. The survival of these relaid mussels is ultimately a function of their quality and physiological condition upon relaying and it has been recognised that mussels can suffer from a loss in condition following transportation. We investigated whether the process of being transported to ongrowing plots had a negative effect on the physiological health and resultant behaviour of mussels by simulating transportation conditions in a controlled experiment. Mussels were kept, out of water, in plastic piping to recreate translocation conditions and further, we tested if depth held in a ship hold (0, 1.5 and 3 m) and length of time emersed (12, 24 and 48 h) affected mussel condition and behaviour. Physiological condition was assessed by quantifying mussel tissue pH and whole tissue glucose, glycogen, succinate and propionate concentrations. The rate of byssogenesis was also quantified to estimate recovery following a period of re-immersion. The depth at which mussels were held did not affect any of the physiological indicators of mussel stress but short-term byssus production was affected. Mussels held at 3 m produced fewer byssus threads during the first 72 h following re-immersion compared with mussels at 0 m (i.e. not buried) suggesting that depth held can impede recovery following transportation. Duration of emersion affected all stress indicators. Specifically, mussels held out of water for 48 h had a reduced physiological condition compared with those emersed for just 12 h. This work has practical implications for the benthic cultivation industry and based on these results we recommend that mussels are held out of water for less than 24 h prior to relaying to ensure physiological health and resultant condition is preserved.

The effects of transportation stress and barnacle fouling on predation rates of starfish (Asterias rubens) on mussels (Mytilus edulis).

The starfish, Asterias rubens, is widely distributed throughout the northern hemisphere and is an important predator on benthic mussel (Mytilus edulis) beds. Whilst several studies have examined how the size of individuals determines this predator–prey relationship, less is known about how the physiological condition of the prey (mussels) and the extent of their fouling may alter these relationships. Such issues are of particular interest to those working within the benthic mussel cultivation industry to inform best management practice and to help minimise losses during the aquaculture process. The potential role of starfish in the removal of epibiotic barnacles from mussels, the presence of which increases processing costs within the industry, is also of interest. We tested whether stressing mussels by aerial exposure for 48 h and whether the extent of barnacle fouling on mussels affected the feeding rates of three different size classes of starfish feeding on two different size classes of mussels. Feeding rates on stressed and unstressed mussels were similar for each starfish–mussel size combination. Barnacle fouling reduced the feeding rate of medium-sized starfish on larger-sized mussels. We also observed starfish, of all size classes, preying directly on the epibiotic barnacles on mussels, however, feeding rates were low and considered unlikely to reduce the extent of fouling on mussels. Our findings show that the predator–prey relationship between starfish and mussels does not differ between unstressed mussels and those experimentally stressed by aerial exposure for 48 h so that this level of stress is unlikely to affect predation rates by A. rubens following relaying in commercial operations. Whilst barnacle fouling suppressed predation rates in one of our experimental treatments, it does not appear that fouling by barnacles would provide a significant refuge from predation for the majority of mussels in benthic aquaculture stocks. Instead we found the size relationship between starfish and mussels was more important in determining predation rates. Starfish are also unlikely to help reduce barnacle fouling on cultured mussels by preying solely on fouling barnacles and the need to control starfish predation during culture remains.

Effects of baited crab pots on cultivated mussel (Mytilus edulis) survival rates

The shore crab, Carcinus maenas, is recognized as a voracious predator of blue mussels, Mytilus edulis, having the potential to greatly reduce stocks in the benthic cultivation industry. As a consequence, baited crab pots are often deployed on and around cultivated benthic mussel beds to trap and remove crabs, in an attempt to reduce predatory pressure. Little is known about how C. maenas behaves around crab pots, but for many other crustacean fisheries, the trapping efficiency of pots is often low. Crabs may be attracted towards but not enter pots, instead feeding on cultivated mussels outside pots on the surrounding substratum. We tested whether the rate of loss of mussels attached to plates differed in areas next to baited pots compared with unbaited pots and to areas without any pots, at two sea loughs (60 km apart) in Northern Ireland. In Strangford Lough, more mussels were lost from plates next to baited pots than the other treatments. In Carlingford Lough, however, we found no difference in the number of mussels lost from plates in any treatment. This difference could be attributed to the different assemblages of mobile benthic predators at the two loughs. The presence of the starfish Asterias rubens, which was absent from experimental sites in Carlingford Lough, was thought to be responsible for increased predation rates near baited pots in Strangford. It is, therefore, important to consider local predator communities when deploying crab pots as a predator mitigation technique to ensure predation rates are in fact reduced and not enhanced. This study is of relevance not only to attempts to limit predation on commercial stocks of benthic cultivated mussels but also in situations where baited traps are deployed close to species vulnerable to mobile benthic predators.

Efficiency of starfish mopping in reducing predation on cultivated benthic mussels (Mytilus edulis Linnaeus)

The starfish, Asterias rubens, preys on mussels (Mytilus edulis), which are relaid during benthic cultivation processes. Starfish mops, a modified dredge used to remove starfish from mussel cultivation beds, are used in several fisheries today but few studies have attempted to quantify the effectiveness of this method in removing starfish. This study tested the effectiveness of starfish mopping to reduce starfish numbers on mussel beds in Belfast Lough, Northern Ireland. Video surveys to determine starfish densities on mussel beds were conducted between October 2013 and December 2014 using a GoPro™ camera attached to starfish mops. This allowed us to firstly test whether starfish density varied among mussel beds and to investigate how fluctuations in starfish numbers may vary in relationship to starfish ecology. We then estimated the efficiency of mops at removing starfish from mussel beds by comparing densities of starfish on beds, as determined using video footage, with densities removed by mops. Starfish abundance was similar among different mussel beds during this study. The efficiency of mops at removing estimated starfish aggregations varied among mussel beds (4–78%) and the mean reduction in starfish abundance was 27% (± SE 3.2). The effectiveness of mops at reducing starfish abundance was shown to decline as the initial density of starfish on mussel beds increased. It can be recommended that the exact deployment technique of mops on mussel beds should vary depending on the density of starfish locally. The area of mussel bed covered by mops during a tow, for example, should be less when starfish densities are high, to maintain efficiencies throughout the full length of tows and to optimise the removal of starfish from mussel beds. This strategy, by reducing abundance of a major predator, could assist in reducing losses in the mussel cultivation industry.

Breaking and Entering: Examining the role of stress and aerial exposure in predator-prey relationships between the common shore crab (Carcinus maenas) and cultivated blue mussels (Mytilus edulis).

During benthic cultivation Mytilus edulis (blue mussels) are subject to predation pressure from a number of predators including Carcinus maenas (shore crabs). This predator can be responsible for substantial losses of mussels from the fishery and a full understanding of the predator–prey relationship between M. edulis and C. maenas is required to ensure attempts that reduce predatory pressure and subsequent commercial loss are successful. Whilst much work has examined the prey–predator size relationships between C. maenas and M. edulis, far less research has investigated how stress, such as periods of extended aerial exposure, may affect these relationships. We tested whether profit in terms of calories gained by crabs consuming mussels stressed by aerial exposure for 48 h differed from that of mussels at ambient conditions and whether being stressed affected the mussel's likelihood of predation. We also tested whether the size relationship between predators and their prey differed when mussels were stressed. We found that the profitability of prey (calories gained per second of handling time) did not vary between stressed and unstressed mussels. Handling times for stressed and unstressed mussels were similar, even when crabs were presented with mussels of the maximum size that they are able to consume. Small crabs were more likely to reject a mussel of preferred size if it was unstressed, suggesting that crabs may be able to assess that these mussels would require extra effort to break into and consume. Our findings suggest that the predator–prey relationship between mussels and crabs is not altered when mussels are stressed. C. maenas remains a voracious predator and regardless of the condition of mussels laid on commercial beds there is a need to control this predator in attempt to reduce losses in the benthic fishery.

Efeitos da acidificação da água do mar na reprodução de Mytilus edulis

Dissertação de mest., Biologia Marinha (Ecologia e Biodiversidade Marinha), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011