Evaluation of Adenovirus and E. coli as indicators for human enteric viruses presence in mussels produced in La Spezia Gulf (Italy)

Aims: The purpose of this work was to verify whether E. coli is a good indicator of viral contamination in mussels and Adenovirus could represent a better alternative as indicator organism of viral presence to guarantee consumer health protection. Methods and Results: Eighty samples of mussels from La Spezia Gulf were analysed for E. coli, Salmonella, Adenovirus, Norovirus and hepatitis A virus with cultural and biomolecular tests. The results of bacterial parameters showed E. coli within the law’s limits and the absence of Salmonella. Twelve samples were positive for Adenovirus presence, one for Norovirus genogroup II and two for hepatitis A virus. None of these positive mussels was found to be contaminated with more than one virus at the same time. Conclusion: This study showed that there was not a direct correlation between the presence of human pathogenic viruses and bacterial indicators. Significance and Impact of the Study: Both E. coli and Adenovirus cannot be considered valid substitutes for the direct research of human pathogenic viruses in mussels. To improve consumer health protection, the European Commission will provide standardized methods for Norovirus and hepatitis A virus detection as soon as possible.     

Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum

We assessed the degree to which Cd, Cr and Zn bound with sediment were assimilated by the green mussel Perna viridis and the Manila clam Ruditapes philippinarum. The influences of the metal concentration in the sediment, the presence of phytoplankton, and the oxidation condition of the sediment on metal assimilation were examined. No major difference was found for metal assimilation efficiency (AE) in sediment with different metal concentrations, except for Cd in the green mussels, in which the AE increased by 1.7x when the Cd concentration in sediment was elevated to 15x the natural background level. The higher assimilation of Cd with increasing Cd load in ingested sediment may be due to the higher desorption of Cd in the acidic gut of the bivalves. Both mussels and clams assimilated metals at a higher efficiency from the diatom diet (Thalassiosira pseudonana) than from inorganic sediment particles. The presence of algal particles had little influence on metal assimilation from ingested sediment, and conversely, the presence of sedimentary particles had little effect on metal assimilation from ingested diatom (except for Cd in the mussels). In the mussels, AEs were higher from oxic sediment than from anoxic sediment by 3.1x for Cd, 2.0x for Cr, and 1.4x for Zn, and in the clams AEs were higher from oxic sediment by 2.8x for Cd, 2.0x for Cr, and 2.0x for Zn. Our study suggested that metals associated with anoxic sediment can be potentially available to marine bivalves, and that metal AEs determined for a single diet were probably not affected by the presence of other food particles.     

Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis

The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.     

Increased gonad ratio in the blue mussel, Mytilus edulis, exposed to starfish predators

The presence of invertebrate predators can induce morphological, physiological, and behavioural changes in sessile marine prey species. In this study, I investigated whether the risk of predation can alter the reproductive effort in the blue mussel Mytilus edulis. In the field, I cultured blue mussels in the presence and in the absence of the starfish, Asterias rubens, using enclosures with two compartments. Two experiments were performed with field periods of 19 and 30 days, respectively. After the field exposure, the mussels were examined for weight of flesh and gonad tissue. In both experiments, the starfish-exposed mussels developed a significantly larger gonad to flesh ratio, compared with that of the unexposed control mussels. After 30 days, the exposed mussels had also lost flesh weight significantly, probably due to spawning. These changes suggest that M. edulis accelerates the gonad development and/or increases its reproductive effort in the presence of predators. Predator-inducible adjustment of life-history characters should be a beneficial trait for this sessile species, that typically encounters a high and variable predation pressure. This revised version was published online in August 2006 with corrections to the Cover Date.     

Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis.

The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.     

Effects of epibiotic algae on the survival, biomass and recruitment of mussels, Mytilus L. (Bivalvia: Mollusca)

Habitats composed of living ‘ecosystem engineers’, such as mussels, are subject to direct and indirect interactions with organisms that live among them. These interactions may affect the presence and structure of habitat, and hence, the associated taxa. We examined the direct effects of epibiotic algae on the survival, biomass and recruitment of mussels (Mytilus L.) on the west coast of Ireland. A field experiment showed that the presence of epibiotic fucoid algae reduced the likelihood of survival of mussels during storms. We also found that the strength of attachment of mussels did not increase in the presence of epibionts. Another in situ experiment revealed that the presence of ephemeral epibiotic algal mats had no effect on the biomass of host mussels, suggesting no effect on mussel growth or production. The abundance of small mussels (< 5 mm) covaried with cover of epibiotic algae at one location, but experimental results suggest no direct effect of the algae on recruitment unless through the action of chemical cues which linger after the removal of algae. We have identified a negative direct interaction between epibiotic algae and mussels on exposed rocky shores, which may often be characterised by bottom-up regulation. It is thought that positive interactions may be more important on more sheltered or sedimentary shores where top-down processes are more likely to dominate.     

Optimal foraging versus shared doom effects: interactive influence of mussel size and epibiosis on predator preference

In the western Baltic Sea, the highly competitive blue mussel Mytilus edulis tends to monopolize shallow water hard substrata. In many habitats, mussel dominance is mainly controlled by the generalist predator Carcinus maenas. These predator-prey interactions seem to be affected by mussel size (relative to crab size) and mussel epibionts.There is a clear relationship between prey size and predator size as suggested by the optimal foraging theory: Each crab size class preferentially preys on a certain mussel size class. Preferred prey size increases with crab size.Epibionts on Mytilus, however, influence this simple pattern of feeding preferences by crabs. When offered similarly sized mussels, crabs prefer Balanus-fouled mussels over clean mussels. There is, however, a hierarchy of factors: the influence of attractive epibiotic barnacles is weaker than the factor ‘mussel size’. Testing small mussels against large mussels, presence or absence of epibiotic barnacles does not significantly alter preferences caused by mussel size. Balanus enhanced crab predation on mussels in two ways: Additional food gain and, probably more important, improvement in handling of the prey. The latter effect is illustrated by the fact that artificial barnacle mimics increased crab predation on mussels to the same extent as do live barnacles.We conclude that crab predation preferences follows the optimal foraging model when prey belong to different size classes, whereas within size classes crab preferences is controlled by epibionts.     

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10-100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.     

THE INFESTATION OF MYTILUS EDULIS LINNAEUS BY POLYDORA CILIATA (JOHNSTON) IN THE CONWY ESTUARY, NORTH WALES

The incidence of the shell-boring parasite Polydora ciliatain Mytilus edulis from the Conwy estuary, North Wales is described.Amongst mussels >40 mm in shell length both the occurrenceand abundance of P. ciliata increased steeply with host size.Infestation was highest amongst mussels near the mouth of theestuary where around 60–65% of the population was parasitized;no systematic relationship between the incidence of P. ciliataand tidal elevation was observed. Mussel condition was negativelycorrelated with the degree of infestation. Moderately and heavilyinfested mussels, however, never accounted for more than ca.10% of this population and the overall detrimental effects ofP. ciliata on growth and production is thus probably quite small.Whilst heavily infested mussels were evidently more vulnerableto predation, the shore crab, Carcinus maenas, selected smaller,non-infested mussels whenever these were available. No obviousrelationships were found between the presence of P. ciliataand the incidence of either pearls or pea crabs. (Received 14 January 1991; accepted 2 April 1991)     

Non-lethal predator effects on the feeding rate and prey selection of the exotic zebra mussel Dreissena polymorpha

Predators may induce changes in prey feeding that indirectly influence both the impact of prey on resource abundances and their interactions with other species in their community. We evaluated whether clearance and excretion (faeces plus pseudofaeces) of phytoplankton by zebra mussels were affected by the presence of predatory cues from roach, Rutilus rutilus, and signal crayfish, Pasifastacus leniusculus . We found that non-lethal effects of predators can alter zebra mussel clearance rate and thus the impact of zebra mussels on phytoplankton. Risk cues released by both predators had similar negative effects on clearance rate of zebra mussels and cascading positive indirect effects on phytoplankton resources. Predation risk had a stronger effect on zebra mussels' clearance rate of cyanobacteria and diatoms than cryptophytes and chrysophytes. The presence of predators did not significantly affect the rate at which zebra mussels expelled and excreted phytoplankton, although there was a tendency for more chlorophyll to be expelled and excreted in the presence of predators. Our results contribute to the growing evidence that predators indirectly affect resource dynamics and food web structure through their non-lethal effects on consumers. Our results suggest that exotic species such as zebra mussels can show behavioural responses to both native (e.g. roach) and exotic (e.g. crayfish) predators.     

Settlement and growth of the green mussel Perna viridis (L.) in coastal waters: influence of water velocity

Green mussels Perna viridis were observed to be a major foulant in the seawater intake tunnel of a coastal power station. Field experiments were carried out to ascertain what factors were responsible for the successful colonisation by mussels. Two adjacent stations (25 m apart) were selected, one representing the coastal waters and the other representing the intake screens (with higher water velocity). Gonadal activity, larval abundance, spat settlement and growth rate of the mussels were monitored at monthly intervals for a total period of two years. The results showed that the breeding activity of the mussels at the study area is influenced largely by temporal distribution of seawater temperature. However, ensuing larval availability in the coastal waters is more dependent on food availability. On the other hand, spat settlement and growth rate are predominantly influenced by water flow, probably as a result of increased propagule and food flux rate at higher water velocities. Higher water velocity at the intake screens also contributed to mussel dominance by preventing settlement of many potential competitors.     

Uptake and retention of Vibrio cholerae non-O1, Salmonella typhi, Escherichia coli and Vibrio harvey by mussels in seawater.

Vibrio cholerae non O1 is known to persist in estuarine and freshwater environments. Experiments evaluated the amount of microorganisms accumulated in mussels maintained in static seawater, contaminated with 10(4) to 10(6) cells/ml and the depuration time required in circulating water. Accumulation and retention times were compared with those for Escherichia coli, Salmonella typhi and Vibrio harvey. E. coli and S. typhi accumulated to a greater extent and were released from mussels more quickly than vibrios which became undetectable 2 to 3 days later than E. coli. Seasonal seawater temperatures (14 to 21 degrees C) had a limited influence on depuration but vibrios appear to be retained with more efficacy over 16 degrees C while E. coli and S. typhi were eliminated to a greater extent. When mussels were contaminated with mixed culture, vibrios appeared to predominate on E. coli, while no interference was observed between E. coli and S. typhi.     

Comparing grazing by Dreissena polymorpha on phytoplankton in the presence of toxic and non-toxic cyanobacteria

SUMMARY 1. The feeding behaviour of the zebra mussel ( Dreissena polymorpha ) was studied in the laboratory on different combinations of food, including a green alga ( Chlamydomonas reinhardtii ) and toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa .
2. The highest clearance rate of phytoplankton by zebra mussels was found when the mussels were feeding on a mixture of Chlamydomonas and non-toxic Microcystis , the lowest on a mixture of Chlamydomonas and toxic Microcystis .
3. The differences found in the clearance rates between food combinations can be partly explained by the production of pseudofaeces containing live phytoplankton cells. Zebra mussels expelled significantly more live phytoplankton cells in the presence of toxic Microcystis than in the presence of non-toxic Microcystis . The pseudofaeces contained predominantly live Chlamydomonas cells. Proportionately much less live Microcystis cells were encountered in the pseudofaeces.
4. Consequently, grazing of zebra mussels on a combination of Chlamydomonas and Microcystis may finally result in a dominance of Chlamydomonas over Microcystis . The presence of toxic Microcystis may even strengthen this shift.     

Oxidative effects of inorganic and organic contaminants on haemolymph of mussels

We applied a newly-established method in haemolymph of mussels, Mytilus galloprovincialis, exposed to different concentrations of heavy metals, such as zinc and cadmium and organic pollutants, such as PAHs and lindane, for the detection of total antioxidant capacity (TAC). The susceptibility of exposed mussels was increased in relation to oxidative stress induced by contaminants tested. Oxidative modifications of proteins were estimated by measuring protein carbonyl content (PCC) and malondialdehyde levels (MDA). For PCC measurement, a highly sensitive and accurate ELISA method, which requires only 5 µg of protein, was used. The significant increase of PCC and MDA in haemolymph of exposed mussels reinforces its role as biomarkers of oxidative stress. Significant correlation of TAC assay, PCC and MDA was conducted in order to evaluate the utility of PCC and TAC assay, used in the present study, as tools for determining oxidative effects of pollutants in mussels. The results reinforce the application of PCC method as useful tool for the determination of PCC alterations in haemolymph of mussels exposed to different levels of contaminants. In addition, the TAC method gives encouraging results, concerning its ability to predict antioxidant efficiency in haemolymph of mussels exposed to inorganic and organic contaminants.     

Factors influencing survival of enterotoxigenic Escherichia coli, Salmonella enterica (serovar Typhimurium) and Vibrio parahaemolyticus in marine environments

The presence and persistence of enterotoxigenic Escherichia coli (ETEC) is poorly investigated in marine habitats. Here we compared ETEC with the more studied fecal contaminant, Salmonella enterica serotype Typhimurium ( S. enterica ) and the marine bacteria Vibrio parahaemolyticus . All three species of bacteria were culturable on agar plates during 8 weeks of incubation in seawater. However, the culturability of ETEC was positively affected by low temperature whereas V. parahaemolyticus was negatively affected. High-nutrient conditions favored the growth of ETEC but not the other bacteria. When the bacteria were fed to blue mussels, V. parahaemolyticus inhibited the filtration activity and the ingestion was lower than that of the enterobacteria. On the other hand, the mussels were less efficient in eliminating V. parahaemolyticus and an in vitro study showed that the hemocytes of three different species of bivalves were not able to kill this strain of V. parahaemolyticus . The bactericidal capacity of bivalves was seemingly an efficient elimination pathway for S. enterica and ETEC. This study showed that ETEC in endemic areas should, to the same degree as S. enterica and V. parahaemolyticus , be taken in consideration when assessing the role of marine environments as a source of enteric infection.     

The Escherichia coli Content of Mytilus edulis from Analysis of Whole Tissue or Digestive Tract

The dissected digestive tract of edible mussels from two sites was examined for Escherichia coli and the counts compared with those from the total tissue of parallel mussel samples. The method using 4 x 1 ml pour plates was preferred to the 1 x 4 ml pour plate or to the 2 x 0.2 ml spread plates. Counts from the digestive tract were up to sixfold higher than parallel counts using total tissue. Digestive tract inoculum also produced amplified Esch. coli detection when lightly polluted mussels were examined by the semi-quantitative percentage-clean technique. It was concluded that dissection of the bacteriologically rich digestive tract significantly increases the sensitivity of Esch. coli detection and has application in environmental assay procedures using mussels or other filter-feeding molluscs. Significant differences in the frequency of Esch. coli biotypes from the two mussel locations could be related to small differences in the pollution regimes at these two sites.     

Potential effects on zooplankton from species shifts in planktivorous mussels: a field experiment in the St Lawrence River

1. Suspension feeding by bivalves exceeds that by other planktivores in many North American rivers, and food webs may be altered substantially by differences in feeding patterns between native unionid mussels and invading dreissenid mussels. 2. We conducted an experiment comparing zooplanktivory by one unionid and two dreissenid species that addressed several primary questions. Is benthic planktivory important in this river? Has this linkage been altered substantially by dreissenids? Do the two dreissenid species differ in planktivory, and is this ecologically important if quagga mussels extend their geographical range? 3. Our 12‐day experiment consisted of controls (no mussels) and treatments with unionid (Elliptio complanata), quagga (Dreissena bugensis) or zebra (D. polymorpha) mussels in 3500‐L, 80‐μm mesh enclosures placed in a slackwater area of the St Lawrence River. 4. The density of the most abundant calanoid copepod Eurytemora affinis increased in the presence of dreissenids, probably as an indirect food web response. By day 12, a cumulative effect was shown by the most overwhelmingly abundant rotifer, Polyarthra, whose density declined dramatically in dreissenid enclosures compared with control and unionid enclosures. Rotifer densities in unionid enclosures were not different from controls, nor were dreissenid treatments different from each other. The effects on rotifers were probably from predation, as Chl‐a did not vary among treatments. 5. We conclude that benthic‐pelagic coupling via planktivory is important in slackwater areas. Dreissenids have strengthened this linkage, but range extension of quaggas should not appreciably alter effects produced by a similar biomass of zebra mussels.     

Factors affecting seasonal mortality of rosy bitterling (Rhodeus ocellatus kurumeus) embryos on the gills of their host mussel

I investigated the seasonal change in factors affecting embryonic mortality in the rosy bitterling, Rhodeus ocellatus kurumeus, a freshwater fish that spawns on the gills of living unionid mussels. Research was conducted in a small pond during 1999 and 2001 in which bitterling were provided with Anodonta sp. mussels for spawning. Bitterling spawned between April and July, peaking mid–late May. Seasonal survival rate of bitterling embryos in their mussel hosts was unimodal, with a peak between late April and mid May (about 70% of total spawnings). In mid April, survival was about 50%. The lowest survival was from late May to July (0%). Losses of bitterling embryos from mussels were identified by ejections from the mussel host. Ejections were categorized as either ejections of live embryos, or ejections of embryos that died in the mussel and were subsequently expelled from the mussel. Ejection rates of live embryos were higher in the earlier part of the spawning period (early–mid April) and dead embryo ejections in the later period (after June). The ejection rate of live embryos was higher among younger embryos earlier in the season, probably because of the incomplete development of morphological and behavioural traits associated with maintaining the embryo inside the mussel gill chambers, and as a consequence of a more protracted developmental period at low temperatures making them more susceptible to ejection. The ejection rate of dead embryos was higher in older embryos later in the season, and in larger mussels and at high embryo densities. The survival of embryos in mussels was probably related to oxygen availability, with mortalities probably caused by asphyxiation. Increased embryo mortalities may arise through competition among embryos, between embryos and mussel, and ambient dissolved oxygen levels. The optimal period for bitterling to spawn may represent a balance between two opposing factors; with positive and negative effects of a seasonal rise in temperature directly affecting embryonic growth rate and oxygen availability.An erratum to this article can be found at This revised version was published online January 2005 with the correction of the authors name.     

Internal shell annuli yield inaccurate growth estimates in the freshwater mussels Elliptio complanata and Lampsilis radiata

1. The objective of this study was to compare, in freshwater bivalves, growth rates inferred from the spacing of internal annuli to those obtained directly by measuring annual changes in shell length.
2. The unionid mussels studied were Elliptio complanata and Lampsilis radiata from a pond in the north-eastern U.S.A. Age was inferred from internal lines of 157 E. complanata and twenty-five L. radiata . Actual length change was determined from comparisons of annual remeasurements of marked E. complanata ( n = 520) in 1992–95 and L. radiata ( n = 81) in 1993–95.
3. High retrieval rates of painted mussels demonstrated the efficacy of the marking method.
4. Annual length changes determined by remeasurement were significantly lower than annual length changes predicted by length-at-age data from internal annuli. Should this be a common occurrence, past estimates of annual growth based on annuli are probably too large, and unionid mussels may be much older than previously assumed.     

Internal shell annuli yield inaccurate growth estimates in the freshwater mussels Elliptio complanata and Lampsilis radiata

1. The objective of this study was to compare, in freshwater bivalves, growth rates inferred from the spacing of internal annuli to those obtained directly by measuring annual changes in shell length.
2. The unionid mussels studied were Elliptio complanata and Lampsilis radiata from a pond in the north-eastern U.S.A. Age was inferred from internal lines of 157 E. complanata and twenty-five L. radiata . Actual length change was determined from comparisons of annual remeasurements of marked E. complanata ( n = 520) in 1992–95 and L. radiata ( n = 81) in 1993–95.
3. High retrieval rates of painted mussels demonstrated the efficacy of the marking method.
4. Annual length changes determined by remeasurement were significantly lower than annual length changes predicted by length-at-age data from internal annuli. Should this be a common occurrence, past estimates of annual growth based on annuli are probably too large, and unionid mussels may be much older than previously assumed.