Impacts of ocean acidification and warming on post-larval growth and metabolism in two populations of the great scallop (Pecten maximus).

Ocean acidification and warming are key stressors for many marine organisms. Some organisms display physiological acclimatization or plasticity, but this may vary across species ranges, especially if populations are adapted to local climatic conditions. Understanding how acclimatization potential varies among populations is therefore important in predicting species responses to climate change. We carried out a common garden experiment to investigate how different populations of the economically important great scallop (Pecten maximus) from France and Norway responded to variation in temperature and PCO2 concentration. After acclimation, post-larval scallops (spat) were reared for 31 days at one of two temperatures (13°C or 19°C) under either ambient or elevated PCO2 (pH 8.0 and pH 7.7). We combined measures of proteomic, metabolic and phenotypic traits to produce an integrative picture of how physiological plasticity varies between the populations. The proteome of French spat showed significant sensitivity to environmental variation, with 12 metabolic, structural and stress-response proteins responding to temperature and/or PCO2. Principal component analysis revealed seven energy metabolism proteins in French spat that were consistent with countering ROS stress under elevated temperature. Oxygen uptake in French spat did not change under elevated temperature but increased under elevated PCO2. In contrast, Norwegian spat reduced oxygen uptake under both elevated temperature and PCO2. Metabolic plasticity allows French scallops to maintain greater energy availability for growth compared with Norwegian spat. However, increased physiological plasticity and growth in French spat may come at a cost, as they showed reduced survival compared with Norwegian scallops under elevated temperature.

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

Transcriptomic analysis of clam extrapallial fluids reveals immunity and cytoskeleton alterations in the first week of Brown Ring Disease development.

The Brown Ring Disease is an infection caused by the bacterium Vibrio tapetis on the Manila clam Ruditapes philippinarum. The process of infection, in the extrapallial fluids (EPFs) of clams, involves alteration of immune functions, in particular on hemocytes which are the cells responsible of phagocytosis. Disorganization of the actin-cytoskeleton in infected clams is a part of what leads to this alteration. This study is the first transcriptomic approach based on collection of extrapallial fluids on living animals experimentally infected by V. tapetis. We performed differential gene expression analysis of EPFs in two experimental treatments (healthy-against infected-clams by V. tapetis), and showed the deregulation of 135 genes. In infected clams, a downregulation of transcripts implied in immune functions (lysosomal activity and complement- and lectin-dependent PRR pathways) was observed during infection. We also showed a deregulation of transcripts encoding proteins involved in the actin cytoskeleton organization such as an overexpression of ß12-Thymosin (which is an actin sequestration protein) or a downregulation of proteins that closely interact with capping proteins such as Coactosin, that counteract action of capping proteins, or Profilin. We validated these transcriptomic results by cellular physiological analyses that showed a decrease of the lysosome amounts and the disorganization of actin cytoskeleton in infected hemocytes.

Deciphering the molecular adaptation of the king scallop (Pecten maximus) to heat stress using transcriptomics and proteomics.

BACKGROUND: The capacity of marine species to survive chronic heat stress underpins their ability to survive warming oceans as a result of climate change. In this study RNA-Seq and 2-DE proteomics were employed to decipher the molecular response of the sub-tidal bivalve Pecten maximus, to elevated temperatures. RESULTS: Individuals were maintained at three different temperatures (15, 21 and 25 °C) for 56 days, representing control conditions, maximum environmental temperature and extreme warming, with individuals sampled at seven time points. The scallops thrived at 21 °C, but suffered a reduction in condition at 25 °C. RNA-Seq analyses produced 26,064 assembled contigs, of which 531 were differentially expressed, with putative annotation assigned to 177 transcripts. The proteomic approach identified 24 differentially expressed proteins, with nine identified by mass spectrometry. Network analysis of these results indicated a pivotal role for GAPDH and AP-1 signalling pathways. Data also suggested a remodelling of the cell structure, as revealed by the differential expression of genes involved in the cytoskeleton and cell membrane and a reduction in DNA repair. They also indicated the diversion of energetic metabolism towards the mobilization of lipid energy reserves to fuel the increased metabolic rate at the higher temperature. CONCLUSIONS: This work provides preliminary insights into the response of P. maximus to chronic heat stress and provides a basis for future studies examining the tipping points and energetic trade-offs of scallop culture in warming oceans.

Factors other than metalloprotease are required for full virulence of French Vibrio tubiashii isolates in oyster larvae.

Vibrio tubiashii is a marine pathogen isolated from larval and juvenile bivalve molluscs that causes bacillary necrosis. Recent studies demonstrated the isolation of this species in a French experimental hatchery/nursery affecting Crassostrea gigas spat in 2007. Here, using larvae of C. gigas as an interaction model, we showed that the French V. tubiashii is virulent to larvae and can cause bacillary necrosis symptoms with an LD50 of about 2.3 × 10(3) c.f.u. ml(-1) after 24 h. Moreover, complete or gel permeation HPLC fractionated extracellular products (ECPs) of this strain appeared toxic to larvae. MS-MS analysis of the different ECP fractions revealed the existence of an extracellular metalloprotease and other suspected virulence factors. This observation is also supported by the expression level of some potential virulence factors. The overall results suggest that the pathology caused by the French V. tubiashii in C. gigas oysters is caused by a group of toxic factors and not only the metalloprotease.

Energy and antioxidant responses of pacific oyster exposed to trace levels of pesticides.

Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 µg/L), isoproturon (0.1 and 1 µg/L), or both in a mixture (0.2 and 0.1 µg/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase α (AMPKα), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was up-regulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.

Transcriptomic response of Enterococcus faecalis V583 to low hydrogen peroxide levels.

Enterococcus faecalis is a Gram-positive commensal bacterium inhabiting the gastrointestinal tracts of human and other mammals, but is also increasingly recognized as an opportunistic human pathogen. Oxidative stress is one of the major challenges encountered by enterococci, both in their natural environment and during infection. In this paper, we evaluated the transcriptomic response of E. faecalis to oxidative stress, and showed that transcript abundance was reduced for 93 genes and increased for 39 genes during growth in medium containing 1.75 mM H2O2. The presence of hydrogen peroxide affected several metabolic pathways, including a large decrease in ethanolamine utilization and methylglyoxal metabolism, and an increase in transcript abundance for several transport systems. In particular, four operons encoding iron transporters appeared highly induced. By contrast, in our experimental conditions, the expression of most of the genes known to be involved in the enterococcal response to oxidative stress, did not appear significantly altered.

Identifying differentially expressed proteins in two-dimensional electrophoresis experiments: inputs from transcriptomics statistical tools.

BACKGROUND: Two-dimensional electrophoresis is a crucial method in proteomics that allows the characterization of proteins' function and expression. This usually implies the identification of proteins that are differentially expressed between two contrasting conditions, for example, healthy versus diseased in human proteomics biomarker discovery and stressful conditions versus control in animal experimentation. The statistical procedures that lead to such identifications are critical steps in the 2-DE analysis workflow. They include a normalization step and a test and probability correction for multiple testing. Statistical issues caused by the high dimensionality of the data and large-scale multiple testing have been a more active topic in transcriptomics than proteomics, especially in microarray analysis. We thus propose to adapt innovative statistical tools developed for microarray analysis and incorporate them in the 2-DE analysis pipeline. RESULTS: In this article, we evaluate the performance of different normalization procedures, different statistical tests and false discovery rate calculation methods with both real and simulated datasets. We demonstrate that the use of statistical procedures adapted from microarrays lead to notable increase in power as well as a minimization of false-positive discovery rate. More specifically, we obtained the best results in terms of reliability and sensibility when using the 'moderate t-test' from Smyth in association with classic false discovery rate from Benjamini and Hochberg. AVAILABILITY: The methods discussed are freely available in the 'prot2D' open source R-package from Bioconductor (http://www.bioconductor.org//) under the terms of the GNU General Public License (version 2 or later). CONTACT: sebastien.artigaud@univ-brest.fr or sebastien.artigaud@gmx.com.

Proteome phenotyping of ΔrelA mutants in Enterococcus faecalis V583.

The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583. A 2-dimensional electrophoresis proteomic analysis of 2 relA mutants, i.e., ΔrelA carrying a complete deletion of the relA gene, and ΔrelAsp that is deleted from only its 3' extremity, showed that 31 proteins were deregulated in 1 or both of these mutants. Mass spectrometry identification of these proteins showed that 10 are related to translation, including 5 ribosomal proteins, 3 proteins involved in translation elongation, and 2 proteins in tRNA synthesis; 14 proteins are involved in diverse metabolisms and biosynthesis (8 in sugar and energy metabolisms, 2 in fatty acid biosynthesis, 2 in amino acid biosynthesis, and 2 in nucleotide metabolism). Five proteins were relevant to the adaptation to different environmental stresses, i.e., SodA and a Dps family protein, 2 cold-shock domain proteins, and Ef1744, which is a general stress protein that plays an important role in the response to ethanol stress. The potential role of these proteins in the development of stress phenotypes associated with these mutations is discussed.

Respiratory response to combined heat and hypoxia in the marine bivalves Pecten maximus and Mytilus spp.

Coastal ecosystems are increasingly disturbed by the increase of mean sea surface temperature and expansion of hypoxic areas. The objectives of the present work were to describe and compare the respiratory responses to combined heat and hypoxia in two bivalve species (Pecten maximus and Mytilus spp.) living in two contrasted coastal habitats (subtidal and intertidal, respectively). Results were consistent with the vertical zonation of both species. Mytilus spp. seemed to cope better with a temperature increase than P. maximus, which was found to be outside of its optimal thermal window at 25°C. Concerning respiratory responses to hypoxia at a given temperature, P. maximus displayed greater oxyregulation capacity that was maintained over a larger range of O2 levels, as compared to Mytilus spp. When acclimation temperatures increased, both species showed a decrease in their oxyregulation capacities alongside a reduction in aerobic performance, especially in P. maximus. The comparison between species suggests that subtidal species, such as P. maximus, might be more vulnerable to a combination of heat and hypoxia than intertidal species, such as Mytilus spp. Lastly, this study highlighted the utility of segmented linear models to estimate PcO2 and regulation percentages in marine organisms exposed to hypoxia.

Relevance of flounder caging and proteomics to explore the impact of a major industrial accident caused by fire on the Seine estuarine water quality.

On September 26th 2019, a major fire occurred in the Lubrizol factory located near the Seine estuary, in Rouen-France. Juvenile flounders were captured in the Canche estuary (a reference system) and caged one month in the Canche and in the Seine downstream the accident site. No significant increases of PAHs, PCBs and PFAS was detected in Seine vs Canche sediments after the accident, but a significant increase of dioxins and furans was observed in water and sewage sludge in the Rouen wastewater treatment plant. The proteomics approach highlighted a dysregulation of proteins associated with cholesterol synthesis and lipid metabolism, in fish caged in the Seine. The overall results suggested that the fire produced air borne dioxins and furans that got deposited on soil and subsequently entered in the Seine estuarine waters via runoff; thus contaminating fish preys and caged flounders in the Seine estuary.

Integration of environmental signatures and omics-based approaches on the European flounder to assist with health assessment of estuarine ecosystems in Brittany, France.

This study aimed to develop a multidisciplinary approach to assess the ecological status of six moderate-sized French estuaries. For each estuary, we gathered geographical information, hydrobiological data, chemistry of pollutants and fish biology, including integration of proteomics and transcriptomics data. This integrative study covered the entire hydrological system studied, from the watershed to the estuary, and considered all the anthropogenic factors that can impact this environment. To reach this goal, European flounder (Platichthys flesus) were collected from six estuaries in September, which ensures a minimum residence time of five months within an estuary. Geographical metrics are used to characterize land use in each watershed. The concentrations of nitrite, nitrate, organic pollutants, and trace elements were measured in water, sediments and biota. All of these environmental parameters allowed to set up a typology of estuaries. Classical fish biomarkers, coupled with molecular data from transcriptomics and shotgun proteomics, highlighted the flounder's responses to stressors in its environment. We analysed the protein abundances and gene expression levels in the liver of fish from the different estuaries. We showed clear positive deregulation of proteins associated with xenobiotic detoxification in a system characterized by a large population density and industrial activity, as well as in a predominantly agricultural catchment area (mostly cultures of vegetables and pig breeding) mainly impacted by pesticides. Fish from the latter estuary also displayed strong deregulation of the urea cycle, most probably related to high nitrogen load. Proteomic and transcriptomic data also revealed a deregulation of proteins and genes related to the response to hypoxia, and a probable endocrine disruption in some estuaries. Coupling these data allowed the precise identification of the main stressors interacting within each hydrosystem.

Physiological and comparative proteomic analyzes reveal immune defense response of the king scallop Pecten maximus in presence of paralytic shellfish toxin (PST) from Alexandrium minutum.

The king scallop, Pecten maximus is a highly valuable seafood in Europe. Over the last few years, its culture has been threatened by toxic microalgae during harmful algal blooms, inducing public health concerns. Indeed, phycotoxins accumulated in bivalves can be harmful for human, especially paralytic shellfish toxins (PST) synthesized by the microalgae Alexandrium minutum. Deleterious effects of these toxic algae on bivalves have also been reported. However, its impact on bivalves such as king scallop is far from being completely understood. This study combined ecophysiological and proteomic analyzes to investigate the early response of juvenile king scallops to a short term exposure to PST producing A. minutum. Our data showed that all along the 2-days exposure to A. minutum, king scallops exhibited transient lower filtration and respiration rates and accumulated PST. Significant inter-individual variability of toxin accumulation potential was observed among individuals. Furthermore, we found that ingestion of toxic algae, correlated to toxin accumulation was driven by two factors: 1/ the time it takes king scallop to recover from filtration inhibition and starts to filtrate again, 2/ the filtration level to which king scallop starts again to filtrate after inhibition. Furthermore, at the end of the 2-day exposure to A. minutum, proteomic analyzes revealed an increase of the killer cell lectin-like receptor B1, involved in adaptative immune response. Proteins involved in detoxification and in metabolism were found in lower amount in A. minutum exposed king scallops. Proteomic data also showed differential accumulation in several structure proteins such as ß-actin, paramyosin and filamin A, suggesting a remodeling of the mantle tissue when king scallops are subjected to an A. minutum exposure.

Impacts of chemical stress, season, and climate change on the flounder population of the highly anthropised Seine estuary (France).

The main objective of this study was to improve our knowledge on the responses of fish populations to multistress (diffuse pollution and warming waters) in estuaries. Adult flounders were caught in two estuaries in the Eastern English Channel: the heavily polluted Seine estuary vs the moderately contaminated Canche estuary. Fish samplings were conducted in January just before the reproduction period, and in July when gonads were at rest. The overall rise in coastal winter water temperatures detected over the Channel impairs the flounder's phenology of reproduction in the two estuaries, inducing a delay of maturation process and probably also spawning. The higher liver histopathology index in Seine vs Canche could be the consequence of the fish exposition to a complex cocktail of contaminants in a strongly industrialized estuary. Higher levels of neurotoxicity, gill lipid peroxidation, and liver EROD activity were observed in Seine vs Canche. Furthermore, a possible impairment in mitochondrial metabolism was suggested in the Seine flounder population. We confirmed in this study the potential role of two membrane lipids (sphingomyelin and phosphatidylserine) in the resistance towards oxidative stress in Seine and Canche. Finally, we suggest that the Seine flounder population (and possibly the connected Eastern English Channel flounder populations over the French Coast) could be seriously impacted in the future by multistress: higher winter temperatures and chemical contamination.

Harsh intertidal environment enhances metabolism and immunity in oyster (Crassostrea gigas) spat.

The Pacific oyster Crassostrea gigas is established in the marine intertidal zone, experiencing rapid and highly dynamic environmental changes throughout the tidal cycle. Depending on the bathymetry, oysters face oxygen deprivation, lack of nutrients, and high changes in temperature during alternation of the cycles of emersion/immersion. Here we showed that intertidal oysters at a bathymetry level of 3 and 5 m delayed by ten days the onset of mortality associated with Pacific Oyster Mortality Syndrome (POMS) as compared to subtidal oysters. Intertidal oysters presented a lower growth but similar energetic reserves to subtidal oysters but induced proteomic changes indicative of a boost in metabolism, inflammation, and innate immunity that may have improved their resistance during infection with the Ostreid herpes virus. Our work highlights that intertidal harsh environmental conditions modify host-pathogen interaction and improve oyster health. This study opens new perspectives on oyster farming for mitigation strategies based on tidal height.

Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum.

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

An Integrated Biomarker Approach Using Flounder to Improve Chemical Risk Assessments in the Heavily Polluted Seine Estuary.

The objective of this study was to develop an integrative approach in ecotoxicology (from biomarkers to population genetics) to assess the ecological status of fish populations. Flounders (Platichthys flesus) collected after the spawning season in the heavily polluted Seine estuary were compared with the moderately polluted Bay of Douarnenez. The muscle energetic reserves were highly depleted in Seine vs. Douarnenez fish. The Seine fish displaying a reduced capacity to manage the oxidative stress and a higher energetic metabolism. An increase in the content of muscle membrane phospholipids (sphingomyelin, phosphatidylserine, free sterols) was detected in the Seine vs. Douarnenez fish. The data integration allowed to hypothesize relationships between membrane phospholipids, xenobiotic metabolism, bioenergetics, and antioxidant defence. The genetic diversity considering neutral markers was maintained in the heavily polluted Seine population compared with the Douarnenez population. Finally, we suggest that the high physiological cost of tolerance to toxicants in the Seine flounder population could compromise its capacity to respond in the future to an additional stressor like warming waters in shallow depth. Thus, this population could be submitted to an ecological risk.

Proteomic analysis and immunogenicity of secreted proteins from Rhodococcus equi ATCC 33701.

Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 degrees C versus 37 degrees C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.

Identification of secreted and surface proteins from Enterococcus faecalis.

Secreted and surface proteins of bacteria are key molecules that interface the cell with the environment. Some of them, corresponding to virulence factors, have already been described for Enterococcus faecalis, the predominant species involved in enterococcal nosocomial infections. In a global proteomic approach, the identification of the most abundant secreted and surface-associated proteins of E. faecalis JH2-2 strain was carried out. These proteins were separated by gel electrophoresis or directly subjected to in vivo trypsinolysis and then analyzed by liquid chromatography - electrospray ion trap tandem mass spectrometry. Putative functions were assigned by homology to the translated genomic database of E. faecalis. A total of 44 proteins were identified, eight secreted proteins from the supernatant culture and 38 cell surface proteins from two-dimensional gel electrophoresis and in vivo trypsinolysis among which two are common to the two groups. Their sequences analysis revealed that 35 of the 44 proteins harbour characteristic features (signal peptide or transmembrane domains) consistent with an extracellular localization. This study may be considered as an important step to encourage proteomic-based investigations of E. faecalis cell surface associated proteins that could lead to the discovery of virulence factors and to the development of new therapeutic tools.

Coupling caging and proteomics on the European flounder (Platichthys flesus) to assess the estuarine water quality at micro scale.

Estuaries are important areas highly vulnerable to anthropogenic pollutions. Therefore, the assessment of estuarine water quality is a major ecological issue. In this study, we sampled juveniles of the European flounder in the "pristine" Canche estuary, and caged them in Canche and in two polluted sites of the Seine estuary, Rouen and Fosse Nord. After one month, the metal and organic pollutants in these sites were assessed, and we evaluated several phenotypic indicators (condition index, RNA/DNA ratios and genotoxicity), and extracted the proteins in fish livers for analysis using a shotgun proteomics approach. The results showed strong modifications in the fish caged in both sites of the Seine estuary, as compared to those caged in Canche. In particular, many proteins involved in phase I and phase II detoxification reactions were accumulated in the liver of fish caged in the site showing the highest pollution, Rouen. In addition, we observed a general disruption of metabolism, in particular an increase in lipid synthesis and carbohydrate degradation in Rouen, and a decrease in the abundance of proteins associated to translational activity in Fosse Nord. At both sites, several stress proteins were decreased. The proteomic impact of the encagement by itself was also evaluated, by comparing the liver proteome of fish caged in Canche to that of fish stayed in natura during the same time. The results showed proteomic signatures of exposure to stressful conditions (particularly heat stress), most probably related to the micro-habitat in which the cages were placed. In conclusion, the caging technique is of great interest for ecotoxicological assessment of estuarine waters, but should consider that the results are representative of the micro-habitat around the cages, which does not necessarily represent the overall heterogeneity of the estuarine environment.