Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components

Marine bivalves are widespread in coastal environments and, due to their filter-feeding habit, they can accumulate large numbers of bacteria thus acting as passive carriers of human pathogens. Bivalves possess both humoral and cellular defence mechanisms that operate in a co-ordinated way to kill and eliminate infecting bacteria. Vibrio species are very abundant in coastal waters and are commonly isolated from edible bivalves tissues where they can persist after depuration processes in controlled waters. Such observations indicate that vibrios are regular components of bivalve microflora and that the molluscs can represent an important ecological niche for these bacteria. Here we tried to summarize data on the interactions between vibrios and bivalve haemolymph; the available evidence supports the hypothesis that persistence of bacteria in bivalve tissues depends, at least in part, on their sensitivity to the bactericidal activity of the haemolymph. Results obtained with an in vitro model of Vibrio cholerae challenged against Mytilus galloprovincialis haemocytes indicate that bacterial surface components, soluble haemolymph factors and the signalling pathways of the haemocyte host are involved in determining the result of vibrio-haemolymph interactions.     

Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, n-CeO(2)) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials.     

Transfection of spermatozoa in bivalve molluscs using naked DNA

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Effects of tumour necrosis factor alpha (TNFalpha) on Mytilus haemocytes: role of stress-activated mitogen-activated protein kinases (MAPKs)

BACKGROUND INFORMATION: Many studies indicate that innate immunity in invertebrates can be modulated by a cytokine network like in vertebrates. In molluscs, the immune response is carried out by circulating haemocytes and soluble haemolymph factors. In the present study, the effects of heterologous TNFalpha (tumour necrosis factor alpha) on cell signalling and function in the haemocytes of the bivalve Mytilus galloprovincialis Lam. were investigated. RESULTS AND CONCLUSIONS: Addition of TNFalpha in the absence of haemolymph serum [in ASW (artificial sea water)] induced cellular stress, as indicated by lysosomal destabilization, and decreased phagocytosis; on the other hand, in the presence of serum, TNFalpha did not affect lysosomal stability and even stimulated phagocytosis. TNFalpha induced rapid phosphorylation of the stress-activated p38 and JNK (c-Jun N-terminal kinase) MAPKs (mitogen-activated protein kinases); both effects were persistent in ASW but transient in serum. Activation of p38 and JNKs in mediating the effects of TNFalpha was confirmed by the use of specific MAPK inhibitors. Moreover, flow cytometric analysis indicated that TNFalpha in the presence of serum induced transient phosphatidylserine exposure on the haemocyte surface, evaluated as annexin V binding; in ASW, the cytokine resulted in a stable increase in the percentage of both annexin- and propidium iodide-positive cells, indicating possible apoptotic/necrotic processes. The results indicate that TNFalpha can affect the function of bivalve haemocytes through conserved transduction pathways involving stress-activated MAPKs and suggest that the haemocyte response to the cytokine is influenced by soluble haemolymph components.     

Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L)

Data are presented that demonstrate the application of the neutral red retention assay (NRR) to monitor the effects of a bacterial inoculation on the haemocyte lysosomes of the European flat oyster Ostrea edulis, Pacific oyster Crassostrea gigas and scallop Pecten maximus. Bivalves were acclimated to three temperature regimes (5, 15 and 25 degrees C), at constant salinity for 7 days in the laboratory. Once baseline responses to acclimation temperature had been established, the effects of an in vivo inoculation on haemocyte lysosomal stability were assessed using the NRR assay. Lysosomal membrane stability was reduced in the presence of bacteria for all three species of bivalve, but destabilisation of C. gigas haemocyte lysosomes appeared to be most sensitive to the presence of the bacterium Listonella anguillarum. For all three bivalve species, the reduction in lysosomal stability appeared to be proportional to the growth of the bacterial inoculate. Using appropriate controls, the NRR assay was demonstrated to have great potential as a tool with which to make rapid initial assessments of the immune status of bivalve molluscs.     

Ecological interactions on macroevolutionary time scales: clams and brachiopods are more than ships that pass in the night

Competition among organisms has ecological and evolutionary consequences. However, whether the consequences of competition are manifested and measureable on macroevolutionary time scales is equivocal. Marine bivalves and brachiopods have overlapping niches such that competition for food and space may occur. Moreover, there is a long‐standing debate over whether bivalves outcompeted brachiopods evolutionarily, because brachiopod diversity declined through time while bivalve diversity increased. To answer this question, we estimate the origination and extinction dynamics of fossil marine bivalve and brachiopod genera from the Ordovician through to the Recent while simultaneously accounting for incomplete sampling. Then, using stochastic differential equations, we assess statistical relationships among diversification and sampling dynamics of brachiopods and bivalves and five paleoenvironmental proxies. None of these potential environmental drivers had any detectable influence on brachiopod or bivalve diversification. In contrast, elevated bivalve extinction rates causally increased brachiopod origination rates, suggesting that bivalves have suppressed brachiopod evolution.     

Age related properties of the Adriatic clam Chamelea gallina (L. 1758) hemocytes

The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.     

侏罗纪双壳类动物群的分布与古气候和古地理因素

生物群的地理分布受各种生物和环境因素的影响,对于不同地史晚期以及不同的生物类型,控制生物群分布的主导因素也不同的。就侏罗纪古大西洋双壳类动物群而言古地理和古气候条件是影响其分布的主要环境因素。     

Interactions between environmental variables determine immunity in the Indian meal moth Plodia interpunctella

1. Animals raised in good environmental conditions are expected to have more resources to invest in immunity than those raised in poor conditions. Variation in immune activity and parasite resistance in response to changes in environmental temperature, population density and food quality have been shown in many invertebrate species. 2. Almost all studies to date have examined the effects of individual variables in isolation. The aim of this study was to address whether environmental factors interact to produce synergistic effects on phenoloxidase (PO) activity and haemocyte count, both indicators of immune system activity. Temperature, food quality and density were varied in a fully factorial design for a total of eight treatment combinations. 3. Strong interactions between the three environmental variables led to the magnitude and in some cases the direction of the effect of most variables changing as the other environmental factors were altered. Overall, food quality had the most important and consistent influence, larvae raised on a good-quality diet having substantially higher PO activity in every case and substantially higher haemocyte counts in all treatments except unheated/low density. 4. When food quality was good, the larvae showed 'density-dependent prophylaxis': raising their investment in immunity when population density is high. When food quality was poor and the temperature low, however, those larvae raised at high densities invested less in immunity. 5. Increased temperature is often thought to lead to increased immune reactivity in ectotherms, but we found that the effect of temperature was strongly dependent on the values of other environmental variables. PO activity increased with temperature when larvae were raised on good food or when density was high, but when food was poor and density low, a higher temperature led to reduced PO activity. A higher temperature led to higher haemocyte counts when density was high and food quality was poor, but in all other cases, the effect of increased temperature was either close to zero or somewhat negative. 6. Although PO activity and haemocyte count were weakly correlated across the whole data set, there were a number of treatments where the two measures responded in different ways to environmental change. Overall, effect sizes for PO activity were substantially higher than those for haemocyte count, indicating that the different components of the immune system vary in their sensitivity to environmental change. 7. Predictions of the effect of environmental or population change on immunity and disease dynamics based on laboratory experiments that only investigate the effects of single variable are likely to be inaccurate or even entirely wrong.     

Tackling intraspecific genetic structure in distribution models better reflects species geographical range

Genetic diversity provides insight into heterogeneous demographic and adaptive history across organisms’ distribution ranges. For this reason, decomposing single species into genetic units may represent a powerful tool to better understand biogeographical patterns as well as improve predictions of the effects of GCC (global climate change) on biodiversity loss. Using 279 georeferenced Iberian accessions, we used classes of three intraspecific genetic units of the annual plant Arabidopsis thaliana obtained from the genetic analyses of nuclear SNPs (single nucleotide polymorphisms), chloroplast SNPs, and the vernalization requirement for flowering. We used SDM (species distribution models), including climate, vegetation, and soil data, at the whole‐species and genetic‐unit levels. We compared model outputs for present environmental conditions and with a particularly severe GCC scenario. SDM accuracy was high for genetic units with smaller distribution ranges. Kernel density plots identified the environmental variables underpinning potential distribution ranges of genetic units. Combinations of environmental variables accounted for potential distribution ranges of genetic units, which shrank dramatically with GCC at almost all levels. Only two genetic clusters increased their potential distribution ranges with GCC. The application of SDM to intraspecific genetic units provides a detailed picture on the biogeographical patterns of distinct genetic groups based on different genetic criteria. Our approach also allowed us to pinpoint the genetic changes, in terms of genetic background and physiological requirements for flowering, that Iberian A. thaliana may experience with a GCC scenario applying SDM to intraspecific genetic units.     

In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes

In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.     

Bivalve aquaculture‐environment interactions in the context of climate change

Coastal embayments are at risk of impacts by climate change drivers such as ocean warming, sea level rise and alteration in precipitation regimes. The response of the ecosystem to these drivers is highly dependent on their magnitude of change, but also on physical characteristics such as bay morphology and river discharge, which play key roles in water residence time and hence estuarine functioning. These considerations are especially relevant for bivalve aquaculture sites, where the cultured biomass can alter ecosystem dynamics. The combination of climate change, physical and aquaculture drivers can result in synergistic/antagonistic and nonlinear processes. A spatially explicit model was constructed to explore effects of the physical environment (bay geomorphic type, freshwater inputs), climate change drivers (sea level, temperature, precipitation) and aquaculture (bivalve species, stock) on ecosystem functioning. A factorial design led to 336 scenarios (48 hydrodynamic × 7 management). Model outcomes suggest that the physical environment controls estuarine functioning given its influence on primary productivity (bottom‐up control dominated by riverine nutrients) and horizontal advection with the open ocean (dominated by bay geomorphic type). The intensity of bivalve aquaculture ultimately determines the bivalve–phytoplankton trophic interaction, which can range from a bottom‐up control triggered by ammonia excretion to a top‐down control via feeding. Results also suggest that temperature is the strongest climate change driver due to its influence on the metabolism of poikilothermic organisms (e.g. zooplankton and bivalves), which ultimately causes a concomitant increase of top‐down pressure on phytoplankton. Given the different thermal tolerance of cultured species, temperature is also critical to sort winners from losers, benefiting Crassostrea virginica over Mytilus edulis under the specific conditions tested in this numerical exercise. In general, it is predicted that bays with large rivers and high exchange with the open ocean will be more resilient under climate change when bivalve aquaculture is present.     

Combined effects of temperature and cadmium exposure on haemocyte apoptosis and cadmium accumulation in the eastern oyster Crassostrea virginica (Gmelin)

Combined effects of acclimation temperature (12, 20 and 28 °C) and exposure to a toxic metal cadmium (Cd, 50 μg L⁻¹) on haemolymph parameters related to immune defense and metal transport were studied in a model marine bivalve, Crassostrea virginica. Acclimation to elevated temperatures resulted in higher plasma protein concentrations and increased Cd levels in oyster haemolymph plasma and haemocytes. Cd accumulation in haemocytes was linear over the 45 days of Cd exposure and accumulation rates were 0.10, 0.53 and 0.56 μg Cd g⁻¹ dry mass at 12, 20 and 28 °C, respectively. Percentage of blood Cd burden associated with haemocytes increased with increasing temperatures from 13–20% at 12 °C to 26–47% at 20 and 28 °C suggesting a higher role for cellular Cd transport at elevated temperatures. Cd levels in gills and hepatopancreas were positively correlated with Cd concentration in haemocytes, but accumulation rates were considerably faster, so that after 45 days of exposure Cd levels in gills and hepatopancreas were >10–20 times higher than in haemocytes. As a result of slow Cd accumulation possibly reflecting fast haemocyte turnover rates and/or exocytosis of Cd-containing granules, haemocytes in Cd-exposed oysters did not reach threshold Cd burdens required to trigger apoptosis. This suggests that haemocyte viability is not likely to contribute to immunosuppression in the environmentally relevant Cd range. In contrast, elevated temperature (28 °C) resulted in a significant increase in the percentage of apoptotic haemocytes compared to 12 or 20 °C supporting the notion that 28 °C is physiologically stressful for C. virginica. Overall, our study demonstrates strong effects of environmental temperature on haemocyte viability and other important blood parameters such as plasma protein content and metal transport capability which may mask potential Cd effects at environmentally relevant exposure levels.     

Fouling deterrence on the bivalve shell Mytilus galloprovincialis: a physical phenomenon?

The physical nature of fouling deterrence by the mussel Mytilus galloprovincialis was investigated using high-resolution biomimics of the bivalve surface. The homogeneous microtextured surface of M. galloprovincialis (1.94 +/- 0.03 microm), the smooth surface of the bivalve Amusium balloti (0 microm), and moulds of these surfaces (biomimics) were compared with controls of smooth (0 microm) and sanded moulds, (55.4 +/- 2.7 microm) and PVC strips (0 microm) in a 12-week field trial. The shell and mould of M. galloprovincialis were fouled by significantly fewer species and had significantly less total fouling cover than the shell and mould of A. balloti over a 12-week period. However, the major effects were between surfaces with and without microtopography. Surface microtopography, be it structured as in the case of M. galloprovincialis shell and mould, or random as in the case of the sanded mould, had a lower cover of fouling organisms than treatments without microtopography after 6 weeks. There was also no difference between the effect of the M. galloprovincialis mould and the sanded mould. The strong fouling deterrent effects of both these surfaces diminished rapidly after 6 to 8 weeks while that of M. galloprovincialis shell remained intact for the duration of the experiment suggesting factors in addition to surface microtopography contribute to fouling deterrence.     

Esterase-like and fibronectin-like polypeptides share similar sex-cell-biased patterns in the gonad of hermaphroditic and gonochoric species of bivalve mollusks

The mechanism of sexualization of the tubular gonad in seawater bivalves is unknown, and no information regarding the genes involved in this process is yet available, except for the identification of esterase (Est)-like "male-associated polypeptide" in the male gonad of Mytilus galloprovincialis. Our present work reveals distinct protein profiles specific for the testicular or ovarian portion of the ovotestis of Pecten maximus. Two proteins exhibiting testis- or ovary-dependent enrichment in the ovotestis have been identified and partially characterized as Est-like and fibronectin (Fn)-like polypeptides, respectively. Immunofluorescence has demonstrated a close association between the localization of these polypeptides and the gonad tubule network and interstitial stroma of the ovotestis of P. maximus. We also present evidence of Est-like and Fn-like protein enrichment, respectively, in testicular and ovarian tissue in hermaphroditic, sex-reversal, and gonochoric species of seawater bivalves. Together, the results (1) strongly suggest that sex-cell-biased expression of Est-like and Fn-like polypeptides in gonad tissue is a widespread phenomenon among bivalve mollusks, despite the high diversification of their sexual patterns, (2) confirm and expand our previous demonstration of sex-biased protein expression in M. galloprovincialis, and (3) indicate a direct link between germ cell differentiation and sexual specialization of the bivalve somatic gonad.     

Spatial and Temporal Examination of Bivalve Communities in Several Estuaries of Southern California and Northern Baja California,MX

A combination of historical bivalve surveys spanning 30–50 years and contemporary sampling were used to document the changes in bivalve community structure over time at four southern California and one northern Baja California estuaries. While there are limitations to the interpretation of historic data, we observed generally similar trends of reduced total bivalve species richness, losses of relatively large and/or deeper-dwelling natives, and gains of relatively small, surface dwelling introduced species across the southern California estuaries, despite fairly distinct bivalve communities. A nearly 50-year absence of bivalves from two wetlands surveyed in a Baja California estuary continued. A combination of site history and current characteristics (e.g., location, depth) likely contributes to maintenance of distinct communities, and both episodic and gradual environmental changes likely contribute to within-estuary temporal shifts (or absences). We highlight future research needed to determine mechanisms underlying patterns so that we can better predict responses of bivalve communities to future scenarios, including climate change and restoration.     

Changes in haemolymph parameters and insect ability to respond to immune challenge during overwintering

Overwintering is a challenging period in the life of temperate insects. A limited energy budget characteristic of this period can result in reduced investment in immune system. Here, we investigated selected physiological and immunological parameters in laboratory‐reared and field‐collected harlequin ladybirds (Harmonia axyridis). For laboratory‐reared beetles, we focused on the effects of winter temperature regime (cold, average, or warm winter) on total haemocyte concentration aiming to investigate potential effects of ongoing climate change on immune system in overwintering insects. We recorded strong reduction in haemocyte concentration during winter; however, there were only limited effects of winter temperature regime on changes in haemocyte concentration in the course of overwintering. For field‐collected beetles, we measured additional parameters, specifically: total protein concentration, antimicrobial activity against Escherichia coli, and haemocyte concentration before and after overwintering. The field experiment did not investigate effects of winter temperature, but focused on changes in inducibility of insect immune system during overwintering, that is, measured parameters were compared between naïve beetles and those challenged by Escherichia coli. Haemocyte concentration decreased during overwintering, but only in individuals challenged by Escherichia coli. Prior to overwintering, the challenged beetles had a significantly higher haemocyte concentration compared to naïve beetles, whereas no difference was observed after overwintering. A similar pattern was observed also for antimicrobial activity against Escherichia coli as challenged beetles outperformed naïve beetles before overwintering, but not after winter. In both sexes, total protein concentration increased in the course of overwintering, but females had a significantly higher total protein concentration in their hemolymph compared to males. In general, our results revealed that insect’s ability to respond to an immune challenge is significantly reduced in the course of overwintering.     

Uptake and Retention of Vibrio parahaemolyticus in a Cohabitating Population of Ruditapes decussatus and Ruditapes philippinarum Under Experimental Conditions

The presence of Vibrio parahaemolyticus in bivalve mollusc is an important cause of foodborne illnesses, and their levels are influenced by environmental changes, such as temperature and salinity. Clams are common species in estuaries and are used in environmental monitoring programmes. Present study compared the uptake and retention of nonpathogenic V. parahaemolyticus by two species of clam (Ruditapes decussatus and R. philippinarum), cohabitating in a closed system. Results showed no significant differences were found between both species of clams. Bacterial levels are following a similar trend with values between 3.48 and 3.70 log CFU/g for R. decussatus and between 3.15 and 3.49 log CFU/g for R. philippinarum. So, in the absence of water renewal, high and stable levels of V. parahaemolyticus were observed in cultured clams after exposure. Changes in physical parameters should be taken into account to design surveillance programmes in bivalves, and sampling should focus on species that have faster filtration rates at that water temperature since they potentially represent the worst-case scenario.