Physiological and molecular responses of the copepods Acartia clausi and Acartia tonsa to nickel nanoparticles and nickel chloride.

Nickel compounds in dissolved form or as nanoparticles may affect planktonic invertebrates in marine ecosystems. Here, we assessed the physiological (naupliar mortality, egg production, egg hatching success) and molecular (quantitative gene expression) responses of the crustacean copepods Acartia clausi (indigenous Mediterranean species) and Acartia tonsa (model organism in ecotoxicology), to nickel nanoparticles (NiNPs) and nickel chloride (NiCl2), over time. We also measured NPs size and the temporal release of Ni ions in aqueous solution, through dynamic light scattering (DLS) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. Nauplii of A. clausi were highly vulnerable to NiCl2 in the 48 h acute test, with an EC50 in the range of Ni concentrations measured in polluted waters. Females of both species exhibited a decreased egg production and hatching success after the 4-day exposure to NiNPs. Molecular responses in A. clausi incubated in NiNPs and NiCl2 showed a stronger up- or down-regulation, compared to A. tonsa, of genes associated with detoxification (phospholipid-hydroperoxide glutathione peroxidase, glutathione-S-transferase sigma), oxidative stress (superoxide dismutase), nervous system functioning (acetylcholinesterase), and oogenesis (vitellogenin) In conclusion, new information was here obtained on the effects of different forms of nickel on physiological and molecular responses of A. clausi, that could help to identify biomarker genes of exposure to be used as early-warning indicators. Our results also highlighted the need of employing indigenous copepod species to better evaluate the ecotoxicological impact of pollutants in different geographical area.

The molecular response of Mytilus coruscus mantle to shell damage under acute acidified sea water revealed by iTRAQ based quantitative proteomic analysis.

Mytilus coruscus is an economically important marine bivalve that lives in estuarine sea areas with seasonal coastal acidification and frequently suffers shell injury in the natural environment. However, the molecular responses and biochemical properties of Mytilus under these conditions are not fully understood. In the present study, we employed tandem mass spectrometry combined with isobaric tagging to identify differentially expressed proteins in the mantle tissue of M. coruscus under different short-term treatments, including shell-complete mussels raised in normal seawater (pH 8.1), shell-damaged mussels raised in normal seawater (pH 8.1), and acidified seawater (pH 7.4). A total of 2694 proteins were identified in the mantle, and analysis of their relative abundance from the three different treatments revealed alterations in the proteins involved in immune regulation, oxidation-reduction processes, protein folding and processing, energy provision, and cytoskeleton. The results obtained by quantitative proteomic analysis of the mantle allowed us to delineate the molecular strategies adopted by M. coruscus in the shell repair process in acidified environments, including an increase in proteins involved in oxidation-reduction processes, protein processing, and cell growth at the expense of proteins involved in immune capacity and energy metabolism. SIGNIFICANCE: The impact of global ocean acidification on calcifying organisms has become a major ecological and environmental problem in the world. Mytilus coruscus is an economically important marine bivalve living in estuary sea area with seasonal coastal acidification, and frequently suffering shell injury in natural environment. Molecular responses of M coruscus under the shell damage and acute acidification is still largely unknown. For this reason, iTRAQ based quantitative proteomic and histological analysis of the mantle from M. coruscus under shell damage and acute acidification were performed, for revealing the proteomic response and possible adaptation mechanism of Mytilus under combined shell damage and acidified sea water, and understanding how the mussel mantle implement a shell-repair process under acidified sea water. Our study provides important data for understanding the shell repair process and proteomic response of Mytilus under ocean acidification, and providing insights into potential adaptation of mussels to future global change.

The Functional Significance of McMafF_G_K in Molluscs: Implications for Nrf2-Mediated Oxidative Stress Response.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal regulator of antioxidant gene expression in mammals, forming heterodimer complexes with small Maf proteins through its BZip domain. However, the underlying mechanism of Nrf2 action in molluscs remains poorly understood. The thick shell mussel, Mytilus coruscus, represents a model organism for the marine environment and molluscs interaction research. In this study, we used in silico cloning to obtain a small Maf homologue called McMafF_G_K from M. coruscus. McMafF_G_K possesses a typical BZip domain, suggesting its affiliation with the traditional small Maf family and its potential involvement in the Nrf2 signaling pathway. Transcriptional analysis revealed that McMafF_G_K exhibited a robust response to benzo[a]pyrene (Bap) in the digestive glands. However, this response was down-regulated upon interference with McMafF_G_K-siRNA. Interestingly, the expression levels of Nrf2, NAD(P)H: quinone oxidoreductase (NQO-1), and Glutathione Peroxidase (GPx), which are key players in oxidative stress response, showed a positive correlation with McMafF_G_K in digested adenocytes of M. coruscus. Furthermore, in vitro analysis of antioxidant capacity in digestive gland cells demonstrated that Bap exposure led to an increase in reactive oxygen species (ROS) levels, accompanied by an elevation in total antioxidant capacity (T-AOC), potentially counterbalancing the excessive ROS. Strikingly, transfection of McMafF_G_K siRNA resulted in a significant rise in ROS level and a down-regulation of T-AOC level. To validate the functional relevance of McMafF_G_K, a glutathione S-transferase (GST) pull-down assay confirmed its interaction with McNrf2, providing compelling evidence of their protein interaction. This study significantly contributes to our understanding of the functional role of McMafF_G_K in the Nrf2 signaling pathway and sheds light on its potential as a target for further research in oxidative stress response.

Transcriptomic response of Mytilus coruscus mantle to acute sea water acidification and shell damage.

Mytilus coruscus is an economically important marine calcifier living in the Yangtze River estuary sea area, where seasonal fluctuations in natural pH occur owing to freshwater input, resulting in a rapid reduction in seawater pH. In addition, Mytilus constantly suffers from shell fracture or injury in the natural environment, and the shell repair mechanisms in mussels have evolved to counteract shell injury. Therefore, we utilized shell-complete and shell-damaged Mytilus coruscus in this study and performed transcriptomic analysis of the mantle to investigate whether the expression of mantle-specific genes can be induced by acute seawater acidification and how the mantle responds to acute acidification during the shell repair process. We found that acute acidification induced more differentially expressed genes than shell damage in the mantle, and the biomineralization-related Gene Ontology terms and KEGG pathways were significantly enriched by these DEGs. Most DEGs were upregulated in enriched pathways, indicating the activation of biomineralization-related processes in the mussel mantle under acute acidification. The expression levels of some shell matrix proteins and antimicrobial peptides increased under acute acidification and/or shell damage, suggesting the molecular modulation of the mantle for the preparation and activation of the shell repairing and anti-infection under adverse environmental conditions. In addition, morphological and microstructural analyses were performed for the mantle edge and shell cross-section, and changes in the mantle secretory capacity and shell inner film system induced by the two stressors were observed. Our findings highlight the adaptation of M. coruscus in estuarine areas with dramatic fluctuations in pH and may prove instrumental in its ability to survive ocean acidification.

Unraveling the protective role of Nrf2 in molluscs: Insights into mitochondrial and apoptosis pathways in the defense against Bap-induced oxidative stress.

Benzopyrene (Bap) is a major constituent of petroleum pollutants commonly found in aquatic environments, and its mutagenic and carcinogenic properties have adverse effects on aquatic organisms' development, growth, and reproduction. The antioxidant defense system element, NF-E2-related factor 2 (Nrf2), has been linked to the oxidative stress response in marine invertebrates exposed to toxic substances. In a previous study, a novel Nrf2 homologue, McNrf2, was identified in mussel Mytilus coruscus, a significant model marine molluscs in ecotoxicology studies. McNrf2 showed the potential to trigger an antioxidant defense against oxidative stress induced by Bap. Here, we employed an Nrf2 overexpression and inhibition model using SFN and ML385 as Nrf2 inducer and inhibitor, respectively. Next, immunofluorescence technique was used to evaluate the nuclear activation of Nrf2 induced by Bap-mediated oxidative stress. Transmission electron microscopy revealed that overexpression of Nrf2 could maintain the quantity and structural integrity of mitochondria, while flow cytometry analysis showed that Nrf2 could alleviate Bap-induced cellular apoptosis. These findings suggest that Nrf2 can protect molluscs from Bap-induced oxidative stress through the mitochondria and apoptosis pathways, providing a novel perspective on Nrf2's antioxidant function.

Metabolic profiling of Mytilus coruscus mantle in response of shell repairing under acute acidification.

Mytilus coruscus is an economically important marine bivalve mollusk found in the Yangtze River estuary, which experiences dramatic pH fluctuations due to seasonal freshwater input and suffer from shell fracture or injury in the natural environment. In this study, we used intact-shell and damaged-shell M. coruscus and performed metabolomic analysis, free amino acids analysis, calcium-positive staining, and intracellular calcium level tests in the mantle to investigate whether the mantle-specific metabolites can be induced by acute sea-water acidification and understand how the mantle responds to acute acidification during the shell repair process. We observed that both shell damage and acute acidification induced alterations in phospholipids, amino acids, nucleotides, organic acids, benzenoids, and their analogs and derivatives. Glycylproline, spicamycin, and 2-aminoheptanoic acid (2-AHA) are explicitly induced by shell damage. Betaine, aspartate, and oxidized glutathione are specifically induced by acute acidification. Our results show different metabolic patterns in the mussel mantle in response to different stressors, which can help elucidate the shell repair process under ocean acidification. furthermore, metabolic processes related to energy supply, cell function, signal transduction, and amino acid synthesis are disturbed by shell damage and/or acute acidification, indicating that both shell damage and acute acidification increased energy consumption, and disturb phospholipid synthesis, osmotic regulation, and redox balance. Free amino acid analysis and enzymatic activity assays partially confirmed our findings, highlighting the adaptation of M. coruscus to dramatic pH fluctuations in the Yangtze River estuary.

Transcriptomic analysis reveals responses to a polluted sediment in the Mediterranean copepod Acartia clausi.

Marine sediments are regarded as sinks for several classes of contaminants. Characterization and effects of sediments on marine biota now require a multidisciplinary approach, which includes chemical and ecotoxicological analyses and molecular biomarkers. Here, a gene expression study was performed to measure the response of adult females of the Mediterranean copepod Acartia clausi to elutriates of polluted sediments (containing high concentrations of polycyclic aromatic hydrocarbons, PAHs, and heavy metals) from an industrial area in the Southern Tyrrhenian Sea (Bagnoli-Coroglio). Functional annotation of the A. clausi transcriptome generated as reference here, showed a good quality of the assembly and great homology with other copepod and crustacean sequences in public databases. This is one of the few available transcriptomic resources for this widespread copepod species of great ecological relevance in temperate coastal areas. Differential expression analysis between females exposed to the elutriate and those in control seawater identified 1000 differentially expressed genes, of which 743 up- and 257 down-regulated. Within the up-regulated genes, the most represented functions were related to proteolysis (lysosomal protease, peptidase, cathepsin), response to stress and detoxification (heat-shock protein, superoxide dismutase, glutathione-S-transferase, cytochrome P450), and cytoskeleton structure (α- and ß-tubulin). Down-regulated genes were mostly involved with ribosome structure (ribosomal proteins) and DNA binding (histone proteins, transcription factors). Overall, these results suggest that processes such as transcription, translation, protein degradation, metabolism of biomolecules, reproduction, and xenobiotic detoxification were altered in the copepod in response to polluted elutriates. In conclusion, our results contribute to gaining information on the transcriptomic responses of copepods to polluted sediments. They will also prompt the selection of genes of interest to be used as biomarkers of exposure to PAHs and heavy metals in molecular toxicology studies on copepods, and in general, in comparative functional genomic studies on marine zooplankton.

Insights into the Response in Digestive Gland of Mytilus coruscus under Heat Stress Using TMT-Based Proteomics.

Ocean warming can cause injury and death in mussels and is believed to be one of the main reasons for extensive die-offs of mussel populations worldwide. However, the biological processes by which mussels respond to heat stress are still unclear. In this study, we conducted an analysis of enzyme activity and TMT-labelled based proteomic in the digestive gland tissue of Mytilus coruscus after exposure to high temperatures. Our results showed that the activities of superoxide dismutase, acid phosphatase, lactate dehydrogenase, and cellular content of lysozyme were significantly changed in response to heat stress. Furthermore, many differentially expressed proteins involved in nutrient digestion and absorption, p53, MAPK, apoptosis, and energy metabolism were activated post-heat stress. These results suggest that M. coruscus can respond to heat stress through the antioxidant system, the immune system, and anaerobic respiration. Additionally, M. coruscus may use fat, leucine, and isoleucine to meet energy requirements under high temperature stress via the TCA cycle pathway. These findings provide a useful reference for further exploration of the response mechanism to heat stress in marine mollusks.

Transcriptome profiling reveals the strategy of thermal tolerance enhancement caused by heat-hardening in Mytilus coruscus.

The thick-shell mussel Mytilus coruscus serves as a common sessile intertidal species and holds economic significance as an aquatic organism. M. coruscus often endure higher temperatures than their ideal range during consecutive low tides in the spring. This exposure to elevated temperatures provides them with a thermal tolerance boost, enabling them to adapt to high-temperature events caused by extreme low tides and adverse weather conditions. This phenomenon is referred to as heat-hardening. Some related studies showed the phenomenon of heat-hardening in sessile intertidal species but not reported at the mechanism level based on transcriptome so far. In this study, physiological experiments, gene family identification and transcriptome sequencing were performed to confirm the thermotolerance enhancement based on heat-hardening and explore the mechanism in M. coruscus. A total of 2935 DEGs were identified and the results of the KEGG enrichment showed that seven heat-hardening relative pathways were enriched, including Toll-like receptor signal pathway, Arachidonic acid metabolism, and others. Then, 24 HSP70 members and 36 CYP2 members, were identified, and the up-regulated members are correlated with increasing thermotolerance. Finally, we concluded that the heat-hardening M. coruscus have a better thermotolerance because of the capability of maintaining the integrity and the phenomenon of vasodilation of the gill under thermal stress. Further, the physiological experiments yielded the same conclusions. Overall, this study confirms the thermotolerance enhancement caused by heat-hardening and reveals the survival strategy in M. coruscus. In addition, the conclusion provides a new reference for studying the intertidal species' heat resistance mechanisms to combat extreme heat events and the strategies for dealing with extreme weather in aquaculture under the global warming trend.

Regulation of innate immunity in marine mussel Mytilus coruscus: MicroRNA Mc-novel_miR_196 targets McTLR-like1 molecule to inhibit inflammatory response and apoptosis.

Toll-like receptors (TLRs) are crucial players in immune recognition and regulation, with aberrant activation leading to autoimmune, chronic inflammatory, and infectious diseases. MicroRNAs (miRNAs) have been shown to regulate gene expression at transcriptional and post-transcriptional levels. While miRNA-mediated regulation of TLR signaling has been studied in mammals, the underlying mechanisms of TLR-miRNA interactions in molluscs remain unclear. In a previous study, one of the TLR genes potentially targeted by miRNAs was identified and named McTLR-like1. McTLR-like1 was later found to be targeted by miRNA Mc-novel_miR_196 through bioinformatic prediction. In this study, we aim to experimentally determine the interaction between McTLR-like1 and Mc-novel_miR_196, as well as their functional role in the innate immune response of molluscs. The results showed that the expression of Mc-novel_miR_196 was suppressed, while the expression of McTLR-like1 was enhanced in M. coruscus hemocytes treated with lipopolysaccharide (LPS). Moreover, in vitro assays demonstrated that Mc-novel_miR_196 directly targets the 5' UTR of McTLR-like1 and leads to the down-regulation of proinflammatory cytokines in hemocytes. In addition, co-transfection experiments confirmed that Mc-novel_miR_196 inhibits McTLR-like1 and inhibits the expression of proinflammatory cytokines. The Tunel assay also showed that Mc-novel_miR_196 inhibited apoptosis in hemocytes induced by LPS. Our findings suggest that microRNA Mc-novel_miR_196 acts as a regulator of innate immunity in M. coruscus by targeting McTLR-like1 and inhibiting inflammatory response and apoptosis. These results provide further insights into the complex molecular mechanisms underlying TLR signaling in molluscs.

Eco-Friendly Engineered Nanomaterials Coupled with Filtering Fine-Mesh Net as a Promising Tool to Remediate Contaminated Freshwater Sludges: An Ecotoxicity Investigation.

The use of eco-friendly engineered nanomaterials represents a recent solution for an effective and safe treatment of contaminated dredging sludge. In this study, an eco-designed engineered material based on cross-linked nanocellulose (CNS) was applied for the first time to decontaminate a real matrix from heavy metals (namely Zn, Ni, Cu, and Fe) and other undesired elements (mainly Ba and As) in a lab-scale study, with the aim to design a safe solution for the remediation of contaminated matrices. Contaminated freshwater sludge was treated with CNS coupled with a filtering fine-mesh net, and the obtained waters were tested for acute and sublethal toxicity. In order to check the safety of the proposed treatment system, toxicity tests were conducted by exposing the bacterium Aliivibrio fischeri and the crustacean Heterocypris incongruens, while subtoxicity biomarkers such as lysosomal membrane stability, genetic, and chromosomal damage assessment were performed on the freshwater bivalve Dreissena polymorpha. Dredging sludge was found to be genotoxic, and such genotoxicity was mitigated by the combined use of CNS and a filtering fine-mesh net. Chemical analyses confirmed the results by highlighting the abetment of target contaminants, indicating the present model as a promising tool in freshwater sludge nanoremediation.

Comparative transcriptomic analysis revealed changes in multiple signaling pathways involved in protein degradation in the digestive gland of Mytilus coruscus during high-temperatures.

As a result of global warming, the Mytilus coruscus living attached in the intertidal zone experience extreme and fluctuating changes in temperature, and extreme temperature changes are causing mass mortality of intertidal species. This study explores the transcriptional response of M. coruscus at different temperatures (18 °C, 26 °C, and 33 °C) and different times (0, 12, and 24 h) of action by analyzing the potential temperature of the intertidal zone. In response to high temperatures, several signaling pathways in M. coruscus, ribosome, endocytosis, endoplasmic reticulum stress, protein degradation, and lysosomes, interact to counter the adverse effects of high temperatures on protein homeostasis. Increased expression of key genes, including heat shock proteins (Hsp70, Hsp20, and Hsp110), Lysosome-associated membrane glycoprotein (LAMP), endoplasmic reticulum chaperone (BiP), and baculoviral IAP repeat-containing protein 7 (BIRC7), may further mitigate the effects of heat stress and delay mortality in M. coruscus. These results reveal changes in multiple signaling pathways involved in protein degradation during high-temperature stress, which will contribute to our overall understanding of the molecular mechanisms underlying the response of M. coruscus to high-temperature stress.

Mytilus farming drives higher local bacterial diversity and facilitates the accumulation of aerobic anoxygenic photoheterotrophic related genera.

Research to assess the impacts of mariculture on the microbiota of the surrounding environment is still inadequate. Here, we examined the effects of Mytilus coruscus farming on the diversity of bacterial community in surrounding seawater using field investigations and indoor simulations, focusing on the variation of members of aerobic anoxygenic photoheterotrophic (AAP) bacteria. In the field, Mytilus farming shaped bacterial community and significantly increased their diversity, including biomass, OTUs, Shannon, relative abundance, number of enriched species, as compared with the non-farming area. Higher abundance of AAP related genera was observed in the Mytilus farming seawater. Under the controlled condition, the presence of M. coruscus significantly shaped the bacterial community composition and caused species composition to become similar after 10 days. Furthermore, the presence of M. coruscus consistently strengthened local diversity in seawater bacterial community, with linkages to the recruitment of AAP members as well. In addition, the tissue-related composition of M. coruscus significantly differed from those in seawater. Our findings highlight a ecological importance of Mytilus farming, as process that shape surrounding water-cultured bacterial community and offer experimental evidence for the accumulation of AAP-related genera in aquaculture systems.

Myticofensin, a novel antimicrobial peptide family identified from Mytilus coruscus.

In this study, seven transcripts representing a novel antimicrobial peptide (AMP) family with structural features similar to those of arthropod defensins were identified from Mytilus coruscus. These novel defensins from the Mytilus AMP family were named myticofensins. To explore the possible immune-related functions of these myticofensins, we examined their expression profiles in different tissues and larval stages, as well as in three immune-related tissues under the threat of different microbes. Our data revealed that the seven myticofensins had relatively high expression levels in immune-related tissues. Most myticofensins were undetectable, or had low expression levels, in different larval mussel stages. Additionally, in vivo microbial challenges significantly increased the expression levels of myticofensins in M. coruscus hemocytes, gills, and digestive glands, showing different immune response patterns under challenges from different microbes. Our data indicates that different myticofensins may have different immune functions in different tissues. Furthermore, peptide sequences corresponding to the beta-hairpin, alpha-helix, and N-terminal loop of myticofensin were synthesized and the antimicrobial activities of these peptide fragments were tested. Our data confirms the diversity of defensins in Mytilus and reports the complex regulation of these defensins in the mussel immune response to different microbes in immune-related tissues. The immune system of Mytilus has been studied for years as they are a species with strong environmental adaptations. Our data can be regarded as a step forward in the study of the adaptation of Mytilus spp. to an evolving microbial world.

Molecular characterization of peptidoglycan recognition proteins from Mytilus coruscus.

Mytilus shows great immune resistance to various bacteria from the living waters, indicating a complex immune recognition mechanism against various microbes. Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Therefore, eight PGRPs were identified from the gill transcriptome of Mytilus coruscus. The sequence features, expression pattern in various organs and larval development stages, and microbes induced expression profiles of these Mytilus PGRPs were determined. Our data revealed the constitutive expression of PGRPs in various organs with relative higher expression level in immune-related organs. The expression of PGRPs is developmentally regulated, and most PGRPs are undetectable in larvae stages. The expression level of most PGRPs was significantly increased with in vivo microbial challenges, showing strong response to Gram-positive strain in gill and digestive gland, strong response to Gram-negative strain in hemocytes, and relative weaker response to fungus in the three tested organs. In addition, the function analysis of the representative recombinant expressed PGRP (rMcPGRP-2) confirmed the antimicrobial and agglutination activities, showing the immune-related importance of PGRP in Mytilus. Our work suggests that Mytilus PGRPs can act as pattern recognition receptors to recognize the invading microorganisms and the antimicrobial effectors during the innate immune response of Mytilus.

Cellular Responses Induced by Zinc in Zebra Mussel Haemocytes. Loss of DNA Integrity as a Cellular Mechanism to Evaluate the Suitability of Nanocellulose-Based Materials in Nanoremediation.

Zinc environmental levels are increasing due to human activities, posing a threat to ecosystems and human health. Therefore, new tools able to remediate Zn contamination in freshwater are highly recommended. Specimens of Dreissena polymorpha (zebra mussel) were exposed for 48 h and 7 days to a wide range of ZnCl2 nominal concentrations (1-10-50-100 mg/L), including those environmentally relevant. Cellulose-based nanosponges (CNS) were also tested to assess their safety and suitability for Zn removal from freshwater. Zebra mussels were exposed to 50 mg/L ZnCl2 alone or incubated with 1.25 g/L of CNS (2 h) and then removed by filtration. The effect of Zn decontamination induced by CNS has been verified by the acute toxicity bioassay Microtox®. DNA primary damage was investigated by the Comet assay; micronuclei frequency and nuclear morphological alterations were assessed by Cytome assay in mussels' haemocytes. The results confirmed the genotoxic effect of ZnCl2 in zebra mussel haemocytes at 48 h and 7-day exposure time. Zinc concentrations were measured in CNS, suggesting that cellulose-based nanosponges were able to remove Zn(II) by reducing its levels in exposure waters and soft tissues of D. polymorpha in agreement with the observed restoration of genetic damage exerted by zinc exposure alone.

Historical control data in ecotoxicology: Eight years of tests with the copepod Acartia tonsa.

The calanoid copepod Acartia tonsa is one of the most frequently used organisms in acute, short-term bioassays to assess effects induced by marine matrices or chemicals on different life stages. Physiological responses in such tests can be highly variable and historical control data (HCD), values recorded from previous studies performed under similar conditions, can be useful to recognise the average responses over time. Here, we analysed egg hatching success and larval (naupliar) immobilisation/mortality of A. tonsa Mediterranean strain, cultured in laboratory conditions since 2008 and used as model organisms in ecotoxicology tests. Our aims were to evaluate the physiological response and sensitivity of A. tonsa over eight years of bioassays, and to compare our HCD with reference values, in order to assess the suitability of such a long-term culture for ecotoxicology studies. Acartia tonsa eggs were exposed for 48 h to the reference toxicant nickel chloride (NiCl2) and the % of egg hatching success and naupliar viability were compared to controls. A total of 59 acute tests, displayed in Shewhart-like control charts, showed a high mean percentage of egg hatching success (85.60% ± 5.90 SD) recorded for the whole period, and a low mean percentage of naupliar immobilisation/mortality (6.73% ± 6.38 SD) in controls. Effective concentration (EC50) for NiCl2 registered a stable mean of 0.14 mg Ni/L (± 0.047 SD) over time. Overall, our long-term dataset confirms the suitability of this copepod species for ecotoxicology studies even after years of culturing in laboratory conditions. It is advisable that other laboratories with long-term datasets made their own control charts, to allow data comparison and to improve test protocols. Considering our HCD, we suggest an EC50 of NiCl2 of 0.14 ± 0.09 mg Ni/L for acute tests with the Mediterranean strain of A. tonsa.

Assessment of the relative sensitivity of the copepods Acartia tonsa and Acartia clausi exposed to sediment-derived elutriates from the Bagnoli-Coroglio industrial area.

The sensitivity of the copepods Acartia tonsa, commonly used in standardized tests for environmental risk assessment and A. clausi, the dominant autochthonous congener species in the Mediterranean Sea, was assessed using sediment-derived elutriates from the industrial area of Bagnoli-Coroglio and nickel chloride as referent toxicant. Acute A. clausi naupliar immobilization test showed EC50 for elutriates E25, E56 and E84 of 23.3%, 80.5% and >100%, respectively, compared to 59.5%, 66.6% and >100% in A. tonsa. In the 7 day sublethal test, a reduction in A. clausi egg production rates was observed in all elutriates, but only in E56 for A. tonsa. Elutriate 56, which contained the highest amount of polycyclic aromatic hydrocarbons, also induced 70% mortality in A. clausi females. Although A. clausi was more sensitive than A. tonsa, the two species had convergent responses to the three elutriates, thus opening the venue for a potential use of A. clausi in standardized ecotoxicity tests.

First molecular evidence of the toxicogenetic effects of copper on sea urchin Paracentrotus lividus embryo development.

Bioassays with sea urchin embryos are widely used to define the environmental quality of marine waters. Anomalies during embryogenesis are generally considered as end-points, whereas a toxigenomic approach, despite it is wide use in other species, is yet in its infancy. In the present study we evaluated toxigenic effects induced by copper on the sea urchin Paracentrotus lividus embryo, combining morphological observations with gene expression analysis. Many anthropogenic activities release copper in the marine environment, with harmful effects on aquatic organisms. In the present study P. lidivus embryos were exposed to different concentrations of copper (24, 36, 48 µg/L) and the activation of fifty specific marker genes, involved in different biological processes (stress, skeletogenesis, development/differentiation, detoxification) was investigated at early blastula, late gastrula and pluteus stage. At blastula stage no morphological anomalies were found, with early down-regulation of genes involved in development/differentiation and a moderate up-regulation of some detoxification genes. At gastrula stage a slight increase in developmental anomalies (up to 19% of malformed embryos) was followed by an increased number of targeted genes belonging to the same two classes, relative to the blastula stage. At pluteus stage morphological anomalies increased in a dose dependent manner. All the analyzed genes were strongly up-regulated, stress and skeletogenic genes showing a "late response" and almost all genes were targeted by copper at all the concentrations tested. The present study represents the first molecular report on the potential negative effect of copper on P. lividus embryos in the environment. Gene expression analysis should be considered as a promising tool for future environmental biomonitoring programs.