Unraveling the interplay between environmental microplastics and salinity stress on Mytilus galloprovincialis larval development: A holistic exploration.

The rise of plastic production has triggered a surge in plastic waste, overwhelming marine ecosystems with microplastics. The effects of climate change, notably changing salinity, have shaped the dynamics of coastal lagoons. Thus, understanding the combined impact of these phenomena on marine organisms becomes increasingly crucial. To address these knowledge gaps, we investigated for the first time the interactive effects of environmental microplastics (EMPs) and increased salinity on the early development of Mytilus galloprovincialis larvae. Morphological assessments using the larval embryotoxicity test revealed larval anomalies and developmental arrests induced by EMPs and increased salinity. Transcriptomic analyses targeting 12 genes involved in oxidative stress, apoptosis, DNA repair, shell formation, and stress proteins were conducted on D-larvae uncovered the potential effects of EMPs on shell biomineralization, highlighting the role of Histidine Rich Glycoproteine (HRG) and tubulin as crucial adaptive mechanisms in Mytilus sp. in response to environmental shifts. Furthermore, we explored oxidative stress and neurotoxicity using biochemical assays. Our findings revealed a potential interaction between EMPs and increased salinity, impacting multiple physiological processes in mussel larvae. Our data contribute to understanding the cumulative effects of emerging anthropogenic pollutants and environmental stressors, emphasizing the need for a holistic approach to assessing their impact on marine ecosystems.

Metal contamination and heat stress impair swimming behavior and acetylcholinesterase activity in embryo-larval stages of the Mediterranean mussel, Mytilus galloprovincialis.

Behavioral parameters are increasingly considered sensitive and early bioindicators of toxicity in aquatic organisms. A video-tracking tool was specifically developed to monitor the swimming behaviour of D-larvae of the Mediterranean mussel, Mytilus galloprovincialis, in controlled laboratory conditions. Both maximum and average swimming speeds and trajectories were recorded. We then investigated the impact of copper and silver with or without a moderate rise of temperature on swimming behavior and acetylcholinesterase (AChE) activity of mussel D-larvae and the possible mechanistic link between both biological responses. Our results showed that copper and/or silver exposure, as well as temperature increase, disrupts the swimming behavior of mussel larvae which could compromise their dispersal and survival. In addition, the combined effect of temperature and metals significantly (p < 0.05) increased AChE activity in mussel larvae. Pearson's correlation analysis was performed and results showed that the AChE activity is positively correlated with maximum speeds (r = 0.71, p < 0.01). This study demonstrates the value of behavioral analyzes of aquatic invertebrates as a sensitive and integrate marker of the effects of stressors.

Natural distribution of pure and hybrid Mytilus sp. along the south Mediterranean and North-east Atlantic coasts and sensitivity of D-larvae stages to temperature increases and metal pollution.

The distribution of the Mediterranean mussel Mytilus galloprovincialis extends more and more northwards in the Atlantic. Crossings are frequently observed with the blue mussel Mytilus edulis along the French and English coasts. The aim of this study is firstlyto identify the co-presence of M. galloprovincialis, M. edulis, and their hybrids in different sites of the Mediterranean and Atlantic coasts, and to provide insights for the thermal tolerance and toxicant susceptibility of Mytilus edulis, Mytilus galloprovincialis and their hybrids. Mussels were collected from the shore at 20 sampling sitesin Europe and Tunisia and identified using Me 15/16 primers targeting the adhesive protein gene sequence. Samples were screened for the presence of Mytilus edulis, Mytilus galloprovincialis, and hybrids alleles using PCR. To get more information on hybrids sensitivities to temperature and metals, freshly fertilized eggs of the two species and their hybrids were reared at four temperatures 18, 20, 22, and 24 °C and exposed to concentrations of Cu, Ag, and a mixture of both metals. Arrests of development and malformations were recorded after 48 h of exposure. The genotypic identification of the two species on 20 sites of the Mediterranean and Atlantic coasts carried out during this study confirms the presence of pure and hybrid species of mussel. Our results highlighted that hybrid larvae from a female of M. galloprovincialis are significantly more tolerant to temperature increases than pure larvae of M. galloprovincialis and pure and hybrid larvae of M. edulis. No significant interspecies-differences of sensitivity were noted for metal exposure alone. However, a co-exposure of larvae to both metal and high temperature highlighted the higher tolerance of hybrid larvae from a female of M. galloprovincialis to both stresses. The overall results could allow the prediction of the future evolution of mussel populations facing environmental changes.

Molecular mechanisms underlying the effects of temperature increase on Mytilus sp. and their hybrids at early larval stages.

The present work aims to investigate the effects of water temperature increase on Mytilus galloprovincilis and Mytilus edulis pure larvae (PG, PE) and their hybrids (HFG, HFE). D-larvae were maintained at 18 °C or exposed to a higher temperature of 22 °C for 48 h. Initially, Embryotoxicity test was evaluated. Second, a transcriptomic analysis using a recently developed microarray platform was applied to determine the main biological processes involved in early life stages responses to temperature increase. Finally, an immunofluorescence investigation was performed to bridge the gap between transcriptomic regulation and the real changes at cellular/tissue levels. Embryotoxicity test revealed a higher sensitivity of M. edulis (PE) D-larvae as well as hybrids from females M. edulis (HFE) to temperature increase, with the highest rate of larval malformations. Transcriptomic results indicated a lack of an adequate heat shock protein (Hsp) response in PE and HFE larvae (the high expression was observed in PG larvae); the differential expression of gene involved in translation, energy metabolism and oxidative stress response may contribute to explain the observed complex alterations in the studied conditions. As revealed by immunohistochemistry, cytoskeleton proteins changes associated with a drastic decrease of Histidine-Rich Glycoprotein (HRG) may elucidate the larval abnormalities in shell development observed for PE and HFE larvae. Overall, the results indicate that each type of pure larva (PG and PE) and their respective female hybrid (HFG and HFE) react similarly to the temperature increase. Our data should be carefully considered in view of the water temperature increase in marine ecosystems and especially for the mussel's species in confluence zones.

Compared responses to copper and increased temperatures of hybrid and pure offspring of two mussel species.

This study investigates the combined effects of increased temperatures and copper on the early live stages of the Mediterranean mussel Mytilus galloprovincialis and the blue mussel Mytilus edulis as well as their hybrids. For this purpose, developmental abnormalities was measured after 48 h of exposure as well as a battery of oxidative stress markers such as, antioxidant enzyme activities, lipid peroxidation measured as malondialdehyde accumulation (MDA) and metallothionein contents (MT) and related gene expression pattern. Embryotoxicity and metal accumulation in tissues of mussel larvae exposed to a sublethal concentration of copper (10 µg/L) along with a slight temperature increase from 18 °C to22 °C were significantly increased after 48 h of exposure. Co-exposure to Cu and elevated temperatures significantly increased the antioxidant enzyme activities termed as, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and being maintained at 22 °C only in pure M. galloprovincialis (PG) larvae and female galloprovincialis x male edulis hybrid larvae (HFG). A significant decrease in mRNA abundance of cat, sod, gst gene transcription levels was showed in the same species. Furthermore, metallothionein accumulation increased significantly in PG D-larvae exposed to copper at 22 °C. The same pattern was observed in term of gene expression of MTs cognates (mt-10 and mt-20). Significant increase of MDA levels in pure M. edulis (PE) larvae and their female M. edulis x male M. galloprovincialis hybrid larvae (HFE) combined with a low MTs content were observed. Overall, this study provides clues about the relatively higher resistance and resilience of M. galloprovincialis species compared to M. edulis species under environmental pollution and future climate change scenarios.

Moderate temperature elevation increase susceptibility of early-life stage of the Mediterranean mussel, Mytilus galloprovincialis to metal-induced genotoxicity.

The present study aims to evaluate the effects of copper and silver alone or along with a moderate temperature increase on embryonic development, DNA integrity and target gene expression levels in early life stages of Mytilus galloprovincialis. For this purpose, upon fertilized embryos were exposed to a sub-lethal concentration of Cu (9.54 µg/L), Ag (2.55 µg/L) and to the mixture of the two metals (Cu (6.67 µg/L) + Ag (1.47 µg/L)) along with a temperature gradient (18, 20 and 22 °C). In all experiments, larvae were exposed to stressors for 48 h except for those designed to DNA damage analysis exposed only for 24 h (before shell formation).Our results showed a significant increase in the percentage of malformed D-larvae (p < 0.05) with increasing temperature and exposure to silver and copper alone or in a mixture. Moreover, metal toxicity increased significantly (p < 0.05) with the temperature rise. Genotoxicity was evaluated using classic and modified with Formamidopyrimidine DNA glycosylase (Fpg) Comet assay. Results suggest that co-exposure to metals and temperature significantly increased DNA damage on mussel larvae with a more accentuated oxidative damage. A significant transcription modulation was observed for genes involved in DNA repair and DNA replication (p53, DNA ligase II and topoisomerase II) when larvae are exposed to a single stressor. However, in the case of multiple stresses, caspase involved in the cell apoptosis pathway was overexpressed. Our study suggests that mussel larvae exposed to a moderate increase in temperature may have a compromised ability to defend against genotoxicity. This is particularly relevant in the context of global warming and thermal pollution.

Early and efficient induction of antioxidant defense system in Mytilus galloprovincialis embryos exposed to metals and heat stress.

The present study aims to elucidate the stress response of early life stages of Mytilus galloprovincialis to the combine effects of selected metals and elevated temperature. For this purpose, we investigated the response of a large panel of oxidative stress markers such as catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) activities and lipid peroxidation (thiobarbituric acid reactive substrates (TBARS) concentration) and metallothionein accumulation (MT) as well as selected gene transcription level and metal accumulation in mussels larvae exposed to a sub-lethal concentration of Cu (9.54µg/L), Ag (2.55µg/L) and mixture of the two metals (Cu (6.67µg/L)+Ag (1.47µg/L)) along with a temperature gradient (18, 20 and 22°C) for 48h. Cu and Ag applied as single or mixture were differentially accumulated in mussel larvae according to the exposure temperature. Sod, cat, gst and mt-10 gene transcription levels showed an important increase in larvae exposed to Cu, Ag or to the mix compared to the control condition at 18°C. The same pattern but with higher induction levels was recorded in larvae co-exposed to metals at 20°C. At 22°C, a significant decrease in mRNA abundance of cat, gst and sod and a significant up-regulation of mts targets (mt10 and mt20) were observed. RESULTS: suggest that co-exposure to metals and moderate elevated temperature (20 and 22°C) significantly increased the antioxidant enzyme activities of catalase (CAT), and glutathione-S-transferase (GST) and caused an increase of metal and metallothionein concentrations. In contrast, no significant change in lipid peroxidation products measured as TBARS content was observed indicating a protective response of anti-oxidative system. This study provides first evidences of the early and efficient protective response of antioxidant defense mechanisms in mussel's early life stages facing in multi stressors situations.

High sensitivity of embryo-larval stage of the Mediterranean mussel, Mytilus galloprovincialis to metal pollution in combination with temperature increase.

The present work aimed to assess the effects of two widespread metallic pollutants, copper and silver, along with environmentally-realistic temperature increases, on embryo-larval development of the Mediterranean mussel Mytilus galloprovincialis. First, mussel embryos upon fertilization were exposed for 48 h to increasing concentrations of Cu (0.5-500 µg/L) and Ag (0.1-100 µg/L) at different temperatures (18, 20, 22 or 24 °C) in order to characterize toxicity of each toxicant at the different tested temperatures. Increasing concentrations of a Cu-Ag mixture were then tested in order to assess the mixture effect at different temperatures (18, 20 or 22 °C). Embryotoxicity was measured after 48 h of exposure (D-larvae stage) considering both the percentage of abnormalities and developmental arrest in D-larvae. The results suggest that the optimum temperature for mussel larvae development is 18 °C (12.65± 1.6% malformations) and beyond 20 °C a steep increase of abnormal larvae was observed up to 100% at 24 °C. Ag was more toxic than Cu with a 50% effective concentration (EC50) at 18 °C of 6.58 µg/L and 17.6 µg/L, respectively. Temperature increased the toxicity of both metals as proved with the EC50 at 20 °C at 3.86 µg/L and 16.28 µg/L for Ag and Cu respectively. Toxic unit calculation suggests additive effects of Cu and Ag in mixture at 18 and 20 °C. These results highlight a possible impairment of M. galloprovincialis reproduction in the Mediterranean Sea in relation to increase of both pollutants and water temperature due to global warming.