Effects of the toxic dinoflagellate Alexandrium catenella on the behaviour and physiology of the blue mussel Mytilus edulis.

The effects of harmful algae on bivalve physiology are complex and involve both physiological and behavioural responses. Studying those responses is essential to better describe and predict their impact on shellfish aquaculture and health risk for humans. In this study we recorded for two months the physiological response of the blue mussel Mytilus edulis from Eastern Canada to a one-week exposure to a paralytic shellfish poisoning producing dinoflagellate strain of Alexandrium catenella, isolated from the St Lawrence estuary, Canada. Mussels in a 'control' treatment were fed continuously with a non-toxic diet, while mussels in a 'starvation' treatment were fed the same non-toxic diet the first week and subsequently starved for seven weeks. Mussels in a 'toxic' treatment received A. catenella for one week before being starved until the end of the experiment. Over a two-month experiment we monitored shell and tissue growth, filtration capacity, respiration rate, byssal attachment strength, valve opening behaviour, and toxin content in tissues. Mussels fed normally on the toxic dinoflagellate and accumulated an average of 51.6 µg STXeq 100 g-1 after one week of exposure. After seven weeks of depuration, about half of the specimen showed levels around 18 µg STXeq 100 g-1. The condition index of exposed mussels ('toxic' treatment) decreased rapidly from the start as compared to mussels that received a one-week non-toxic diet ('starvation' treatment). Oxygen consumption rates increased in the 'toxic' treatment before leveling out with that of mussels from the 'starvation' treatment. Valve opening amplitude was lower in the 'toxic' treatment during and following the exposure. Average valve closure duration was higher right after the exposure, during the peak of mussel tissue intoxication. No significant change in byssal thread strength was observed through time in each treatment but less force was required to detach mussels from the 'toxic' and 'starvation' treatments. The number of byssus threads produced by mussels exposed to the toxic dinoflagellate was also lower than in the control group. These results represent advancements in our understanding of the impacts of harmful algae on bivalves and contribute to the development of mitigation measures necessary to both the safety of consumers and the sustainability of aquaculture operations.

The Mediterranean mussel Mytilus galloprovincialis: responses to climate change scenarios as a function of the original habitat.

The impact of simulated seawater acidification and warming conditions on specimens of the mussel Mytilus galloprovincialis locally adapted to very distinct, widely separated sites in the Mediterranean Sea (Tunisia) and Atlantic Sea (Galicia, NW Spain) was evaluated in relation to key behavioural and eco-physiological parameters. Over the 2-month exposure to the experimental conditions, mussels were fed optimally to ensure that there are no synergistic interactions between climate change drivers and energetic status of the individuals. In general, regardless of origin (Atlantic or Mediterranean), the mussels were rather resilient to acidification for most of the parameters considered and they were able to grow in strongly acidified seawater through an increased feeding activity. However, shell strength decreased (40%) consistently in both mussel populations held in moderately and highly acidified seawater. The observed reduction in shell strength was not explained by slight alterations in organic matter, shell thickness or aragonite:calcite ratio. The combined effects of high acidification and warming on the key response of byssus strength caused a strong decline in mussel performance, although only in Galician mussels, in which the valve opening time decreased sharply as well as condition index (soft tissue state) and shell growth. By contrast, the observed negative effect of highly acidified scenario on the strength of Tunisian mussel shells was (partly but not totally) counterbalanced by the higher seawater temperature. Eco-physiological and behavioural interactions in mussels in relation to climate change are complex, and future scenarios for the ecology of the species and also the feasibility of cultivating them in Atlantic and Mediterranean zones are discussed.

Behavioural responses to predators in Mediterranean mussels (Mytilus galloprovincialis) are unaffected by elevated pCO2.

Ocean acidification is expected to affect marine organisms in the near future. Furthermore, abrupt short-term fluctuations in seawater pCO2 characteristic of near-shore coastal regions and high-density aquaculture sites currently have the potential to influence organismal and community functioning by altering animal behaviour. While anti-predator responses in fishes exposed to elevated pCO2 are well documented, such responses in benthic marine invertebrates are poorly studied. We used high frequency, non-invasive biosensors to test whether or not short term (3-week) exposure to elevated pCO2 could impact behavioural responses to the threat of predation in adult Mediterranean mussels from Galicia on the northwestern coast of Spain. Predator alarm cues (crushed conspecifics) resulted in a prolonged (1 h) reduction in the degree of valve opening (-20%) but had no clear effect on overall valve movement activity, while elevated pCO2 did not affect either response. Our results add to the increasing body of evidence suggesting that the effects of end-of-century pCO2 levels on marine animal behaviour are likely weak. Nonetheless, longer-term exposures spanning multiple generations are needed to better understand how ocean acidification might impact behavioural responses to predation in marine bivalves.

The impact of the sea anemone Actinothoe sphyrodeta on Mytilus galloprovincialis mussel cultivation (Galicia, Spain).

Marine mussel aggregations act as a substratum and refuge for many fouling species. Mussel cultivation in Galicia, Spain, is carried out on hanging ropes in subtidal systems. The fauna associated with this cultivation includes a large number of invertebrates that compete for space or food with the mussels, or use their clusters as a refuge from predators or water turbulence. Outbreaks of the epibiont anemone Actinothoe sphyrodeta have been reported in cultivated Galician mussels since 2013, but their impact has not been investigated rigorously. Here, the temporal and spatial variability of Actinothoe sphyrodeta on mussel shells throughout one year is presented. Sampling of mussel size, weight and byssus attachment strength allowed mussel tenacity (attachment strength relative to size) to be calculated. A higher presence of Actinothoe sphyrodeta correlated with lower mussel tenacity and greater biomass losses, suggesting that this species could be an economically important biofouling component.

Interspecies comparison of the mechanical properties and biochemical composition of byssal threads.

Several bivalve species produce byssus threads to provide attachment to substrates, with mechanical properties highly variable among species. Here, we examined the distal section of byssal threads produced by a range of bivalve species (Mytilus edulis, Mytilus trossulus, Mytilus galloprovincialis, Mytilus californianus, Pinna nobilis, Perna perna, Xenostrobus securis, Brachidontes solisianus and Isognomon bicolor) collected from different nearshore environments. Morphological and mechanical properties were measured, and biochemical analyses were performed. Multivariate redundancy analyses on mechanical properties revealed that byssal threads of M. californianus, M. galloprovincialis and P. nobilis have very distinct mechanical behaviours compared with the remaining species. Extensibility, strength and force were the main variables separating these species groups, which were highest for M. californianus and lowest for P. nobilis Furthermore, the analysis of the amino acid composition revealed that I. bicolor and P. nobilis threads are significantly different from the other species, suggesting a different underlying structural strategy. Determination of metal contents showed that the individual concentration of inorganic elements varies, but that the dominant elements are conserved between species. Altogether, this bivalve species comparison suggests some molecular bases for the biomechanical characteristics of byssal fibres that may reflect phylogenetic limitations.

Response of Two Mytilids to a Heatwave: The Complex Interplay of Physiology, Behaviour and Ecological Interactions.

Different combinations of behavioural and physiological responses may play a crucial role in the ecological success of species, notably in the context of biological invasions. The invasive mussel Xenostrobus securis has successfully colonised the inner part of the Galician Rias Baixas (NW Spain), where it co-occurs with the commercially-important mussel Mytilus galloprovincialis. This study investigated the effect of a heatwave on the physiological and behavioural responses in monospecific or mixed aggregations of these species. In a mesocosm experiment, mussels were exposed to simulated tidal cycles and similar temperature conditions to those experienced in the field during a heat-wave that occurred in the summer of 2013, when field robo-mussels registered temperatures up to 44.5°C at low tide. The overall responses to stress differed markedly between the two species. In monospecific aggregations M. galloprovincialis was more vulnerable than X. securis to heat exposure during emersion. However, in mixed aggregations, the presence of the invader was associated with lower mortality in M. galloprovincialis. The greater sensitivity of M. galloprovincialis to heat exposure was reflected in a higher mortality level, greater induction of Hsp70 protein and higher rates of respiration and gaping activity, which were accompanied by a lower heart rate (bradycardia). The findings show that the invader enhanced the physiological performance of M. galloprovincialis, highlighting the importance of species interactions in regulating responses to environmental stress. Understanding the complex interactions between ecological factors and physiological and behavioural responses of closely-related species is essential for predicting the impacts of invasions in the context of future climate change.

From broad-spectrum biocides to quorum sensing disruptors and mussel repellents: antifouling profile of alkyl triphenylphosphonium salts.

'Onium' compounds, including ammonium and phosphonium salts, have been employed as antiseptics and disinfectants. These cationic biocides have been incorporated into multiple materials, principally to avoid bacterial attachment. In this work, we selected 20 alkyl-triphenylphosphonium salts, differing mainly in the length and functionalization of their alkyl chains, in fulfilment of two main objectives: 1) to provide a comprehensive evaluation of the antifouling profile of these molecules with relevant marine fouling organisms; and 2) to shed new light on their potential applications, beyond their classic use as broad-spectrum biocides. In this regard, we demonstrate for the first time that these compounds are also able to act as non-toxic quorum sensing disruptors in two different bacterial models (Chromobacterium violaceum and Vibrio harveyi) as well as repellents in the mussel Mytilus galloprovincialis. In addition, their inhibitory activity on a fouling-relevant enzymatic model (tyrosinase) is characterized. An analysis of the structure-activity relationships of these compounds for antifouling purposes is provided, which may result useful in the design of targeted antifouling solutions with these molecules. Altogether, the findings reported herein provide a different perspective on the biological activities of phosphonium compounds that is particularly focused on, but, as the reader will realize, is not limited to their use as antifouling agents.

Byssus attachment strength of two mytilids in mono-specific and mixed-species mussel beds.

The mussel Xenostrobus securis is endemic to the brackish waters of New Zealand and Australia, but has successfully invaded the inner Galician Rías of NW Spain, where it coexists with the indigenous mussel Mytilus galloprovincialis. In this laboratory study, the plasticity of the byssus attachment strength of two mytilids was compared by manipulating substratum, salinity, and bed assembly. M. galloprovincialis showed stronger byssus detachment strength than X. securis, despite lower byssus coverage. Both species responded similarly to the substratum, with substantially lower byssus strength on methacrylate, which offered the lowest surface free energy. Byssus detachment values for M. galloprovincialis were lower at lower salinity. In mixed beds, a number of mussels moved upwards, eventually colonising the upper layers of the assemblage. This behaviour increased byssus strength but only for X. securis. X. securis is adapted to a wide spectrum of abiotic conditions, a trait that may promote its dissemination within estuarine environments.

Secretion of byssal threads in Mytilus galloprovincialis: quantitative and qualitative values after spawning stress.

The effect of spawning events of the mussel Mytilus galloprovincialis on both quantitative and qualitative values of byssus secretion and its associated attachment force was investigated. Byssogenesis rates and absorption efficiency values were significantly reduced after spawning of individuals. However, the maintenance of individuals under sub-optimal conditions (lack of microalgae in the diet) for a week caused no effect on thread's number. Surprisingly, the attachment force varied within a narrow range of values (1.7-1.9 N) with the exception of a significant drop in the experimental group spawned and kept unfed (1.0 N; P < 0.001), most likely due to a similar pattern of the thread's thickness variability. Qualitative analysis concerned to the amino acid composition of the byssus highlighted a higher presence of the basic residues histidine and lysine in threads secreted by spawned individuals. The presence of both histidine and lysine residues in the byssal collagen is associated to the formation of cross-links and specifically histidine has a functionality with a pronounced effect on metal chelation to stabilise the integrity of the byssus. Results reported here evidence the necessity to integrate all components that eventually determines the attachment strength of the mussels to get more insight to the plasticity of such secretion. Morphology of the byssus (thickness) secreted under different endogenous conditions of mussels was the major parameter to explain variability in the attachment force. Moreover, aminoacidic composition as quality term of the byssus secreted may also contribute to understand the plasticity of this secretion and needs to be extended in further surveys.

Anaerobic survival potential of four bivalves from different habitats. A comparative survey.

A comparative survey of the anaerobic survival potential of four different bivalve species and the interference of associated bacteria has been carried out. Individuals from both subtidal and intertidal environments were considered by selecting the following species: Mytilus edulis (subtidal epifaunal), Spisula subtruncata (subtidal infaunal), Macoma balthica (intertidal infaunal) and Cerastoderma edule (intertidal infaunal). Anaerobiosis was simulated in the laboratory by subjecting individuals to the following conditions: nitrogen atmosphere, air atmosphere and anoxic seawater incubation. Moreover, the effect of the antibiotic CA (chloramphenicol) was investigated, either as a pre-treatment of individuals kept under normoxic conditions for a week or directly added to the anoxic incubation media. According to survival performances of the individuals, intertidal animals that use to cope with tidal fluctuations in the coastline (emersion processes) had an extraordinary greater capacity to survive aerial exposure as compared to both nitrogen gas and anoxic seawater incubations most likely due to their capacity to perform aerobiosis at certain rate from atmospheric oxygen availability. Specifically, Macoma balthica enlarged its survival potential up to 24.8 days (LT(50)) under air exposure at 12 degrees C as compared to other specific treatments used here (4.9 days). The latter pattern was also observed, although in a much lower magnitude, for the other intertidal species Cerastoderma edule that survived 3.7 and 4.6 days (LT(50)) under nitrogen atmosphere and anoxic seawater incubation, respectively as compared to 9.5 days for emersed individuals. In contrast to the subtidal species, aerial exposure of both intertidal species led to a much higher survival performances than incubation of individuals in anoxic media with the presence of antibiotic. Survival capacity of the subtidal species Mytilus edulis and Spisula subtruncata was statistically similar under air and nitrogen atmospheres and anoxic seawater incubation. Then, subtidal species have a limited ability to air breathing as a conclusion of a similar survival in atmospheric and anoxic seawater incubations. Remarkably, M. edulis represented the only exception when considering longer-term survival capacity compared to the LT(50) values. Indeed, differences in LT(90) values for M. edulis were statistically different, values decreasing significantly from 19.7-19.9 days (under both nitrogen and air atmospheres) to 16.7 days when individuals are incubated in anoxic seawater. This may be due to the adverse effects of anaerobic bacteria that spontaneously proliferate within the static seawater incubations. As well as for S. subtruncata, possible aerobic processes under aerial exposure of mussels seemed to be not significant for the enlargement of its survival potential, since results obtained for both air and nitrogen atmospheres are similar. Pre-treatment with the antibiotic chloramphenicol caused survival capacity to increase by a factor of approx. 2 (M. edulis) and 34-44% (S. subtruncata). In contrast to intertidal species, the direct addition of the antibiotic to the incubation media caused the highest survival performances in both subtidal species. Habitat differences and species-dependent variability must be considered as significant sources of variation when studying the anaerobic performance of individuals using the most common experimental anaerobic techniques to test survival potential.

Free amino acid composition in juveniles of Mytilus galloprovincialis: spatial variability after Prestige oil spill.

Composition of free amino acids (FAA) in juveniles of Mytilus galloprovincialis was analysed along a large geographical coastline area in Galicia (NW Spain). Individuals were sampled in February 2003, three months after the Prestige oil spill. Pollution values at sampling time were reported as polycyclic aromatic hydrocarbons (PAHs) concentrations in soft tissues of individuals and varied between the highest amount observed in Carrumeiro mussels (502 ng/g dw) and the lowest in Pindo mussels (196 ng/g dw), both locations being close to each other in the centre of the geographical area under study. Pollution values in the other populations varied within the range of 241-347 ng/g dw. Total free amino acids (TFAA) were highest in Aguiño-Pindo-Carrumeiro juveniles at the centre of the Coastline area studied (420-462 micromol/g dw) as compared to the other populations at North and South of Galicia (312-347 micromol/g dw). TFAA results were based on the variability observed in protein free amino acids (PFAA micromol/g dw) among populations (214-249 micromol/g dw for Aguiño-Pindo-Carrumeiro mussels and 98-149 micromol/g dw for the other populations) whereas non-protein free amino acids (NPFAA) taurine and ornithine did not show any significant spatial pattern of variation. Glycine and alanine represented the most abundant PFAA (16-29% and 2.7-11.9% of TPFAA, respectively) and significant correlations between PFAA and both the protein content of soft tissues (r=-0.82) and the condition index of juveniles (r=0.86) were observed. No significant relationships were detected, however, between pollution values in soft tissues as PAHs and FAA profiles with the exception of alanine concentrations as percentage of TFAA (r=0.88; P<0.01). The latter seemed to be an "all or nothing" effect likely due to the influence of other abiotic factors at one of the sampling sites. Such relationship was found not significant when the outlier represented by Carrumeiro mussels was removed from the analysis. The most abundant free amino acid taurine (43.2-68.5%TFAA) followed an inverse variability of that of glycine and by extension of the group PFAA most likely as a compensatory decrease in mussel populations with low protein content (and high condition index). Accordingly, taurine:glycine (t:g) ratio varied between 1 and 2 in most mussel populations but increased up to 3.2-4.2 in Miranda and Bueu mussels at both ends of the geographical interval studied with a corresponding PAHs concentrations of 261 and 304 ng/g dw, respectively. These mussel populations with the highest t:g ratios were characterised by the lowest PFAA contents (below 40%) and condition index values (below 10%). Results of the present study established a significant link between energetic status of growing juveniles and FAA concentrations in environments with different pollution degrees. Variability of the free amino acids profiles in soft tissues were related to endogenous factors of juveniles (protein content, condition index) whereas no relationship with contamination values could be observed. The utility of t:g ratio as general condition factor for M. galloprovincialis is also corroborated for in situ growing juveniles.

Variability of taurine concentrations in Mytilus galloprovincialis as a function of body size and specific tissue.

Variability of taurine (2-aminoethane sulfonic acid) was studied as a function of size in the mussel Mytilus galloprovincialis and tissue specificity. Isometric and/or allometric relationships were established with regard to total soft mass of the mussels between 20 and 60 mm shell length. Relative amounts of taurine dropped significantly with increasing mass of whole soft tissues with an allometric coefficient value of -0.15. The inverse relationship between taurine and increasing size of mussels was confirmed for gill epithelium and labial palp (allometric coefficient values of -0.16 and -0.10, respectively), tissues that, in turn, represented isometric functions with regard to total soft mass. Although relative amounts of taurine were significantly different in digestive gland, mantle and foot, relationships with increasing size of mussels remained unchanged in these tissues. Gill area of mussels was related to soft mass with an allometric coefficient of 0.70 by 2D Image Analysis, but increased to 0.85 when introducing a third dimension, i.e. gill thickness. Results are discussed according to gill structure analysis and taurine functionality.

Anoxic survival potential of bivalves: (arte)facts.

The anoxic survival time of the bivalves Chamelea gallina, Cerastoderma edule and Scapharca inaequivalvis from two different ecosystems and differing anoxia tolerances was studied in static (closed) and flow-through systems. The antibiotics chloramphenicol, penicillin and polymyxin were added, and molybdate (specific inhibitor of the process of sulfate reduction). Survival in (near) anoxic seawater of Chamelea was studied in a static system by comparing untreated seawater with autoclaved seawater and untreated clams with clams incubated in well-aerated seawater, containing the broad-spectrum antibiotic chloramphenicol, prior to the anoxic survival test. With untreated clams and natural seawater (median mortality time 2.4 days) a decrease in pH and exponential accumulation of sulfide and ammonium was observed in the anoxic medium, indicating excessive growth of (sulfate reducing) bacteria. In sterilized seawater LT50 (2.1 days) was not significantly different and again considerable amounts of ammonium and sulfide accumulated. However, pre-treatment of clams with chloramphenicol resulted in an increase of LT50 (11.0 days) by approximately fivefold. Accumulation of ammonium and sulfide was retarded, but was finally even stronger than in the medium containing untreated clams. Median mortality times were 2.5 and 2.4 days for Chamelea and 2.7 and 2.9 days for Cerastoderma for static and flow-through incubations, respectively. Addition of chloramphenicol increased strongly survival time in both systems with corresponding values of 11.0 and 16.3 days for Chamelea, and 6.4 and 6.5 days for Cerastoderma. LT50 of Scapharca in anoxic seawater was 14.4 days. Chloramphenicol and penicillin increased median survival time to 28.5 and 28.7 days, respectively, whereas polymyxin displayed no effect (LT50=13.6 days). Molybdate added to artificial sulfate free seawater blocked biotic sulfide formation, but did not improve survival time (LT50=13.7 days). Overall the results indicate that proliferation of anaerobic pathogenic bacteria, firmly associated with the bivalves, is a main cause of death besides lack of oxygen. Bacterial damage is probably caused by injury of the tissues of the clams and not by the release of noxious compounds to the medium.