Hepatopancreas Research Articles

Oxidative stress, histopathological and genotoxicity of copper oxide nanoparticles in Biomphalaria alexandrina snail

AbstractHigher usage of copper oxide nanomaterials in industrial and biomedical fields may cause an increase of these nanoparticles in aquatic environments, which could have a detrimental ecological effect. Thus, the objective of this study was to evaluate the acute toxicity of copper oxide nanoparticles on the freshwater gastropod, Biomphalaria alexandrina. Transmission electron microscopy, x-ray diffraction analysis and UV–VIS spectrophotometer of CuO NPs revealed a typical TEM image and a single crystal structure with average crystallite size of approximately 40 nm also, a sharp absorption band was appeared. Following exposure to sub-lethal concentrations of CuO NPs (LC10, 15.6 mg/l and LC25, 27.2 mg/l), treated snails revealed a significant decrease (p < 0.05) in total antioxidant capacity, reduced glutathione contents as well as catalase, and sodium dismutase activities were significantly declined (p < 0.05) in comparison to the control group. Also, histopathological alterations were observed in the digestive gland, including ruptured and vacuolated digestive cells, and a marked increase in the number of secretory cells and the severity of the damage increased with rising concentrations. Furthermore, changes in RAPD profiles were detected in the treated snails. In conclusion, our research highlights the potential ecological impact of CuO NPs release in aquatic ecosystems and advocates for improved monitoring and regulation of CuO NPs industrial usage and disposal.

Paddy straw biochar amended sediment enhances pond productivity, growth and physiological profile of Penaeus vannamei cultured in inland saline nursery ponds.

The growing interest in commercial Inland saline aquaculture has taken momentum across the globe due to the available technologies for aquaculture and the abundant resources of saline groundwater. However, the critical problems in inland saline ponds are degraded soil and imbalanced or deficient nutrients. To address these issues, a 75-day experiment was conducted to explore the effects of Paddy Straw Biochar (PSB) as a sediment amendment on sediment quality, water characteristics, growth parameters, and the well-being of Penaeus vannamei reared in inland saline environments. PSB was applied to the nursery ponds at 1 ton/hectare (Treatment, T1) and 2 ton/hectare (Treatment, T2). The findings of water quality parameters showed a significant increase in K+ and Mg++ with a decrease in ammonia-N levels in biochar-applied ponds. Similarly, the addition of biochar to ponds (T2) resulted in improved sediment characteristics with enhanced water holding capacity (24.75%), increased soil organic carbon content (77.94%), pH levels (0.37 units), cation exchange capacity (43.15%), available potassium (16.3%), and lower bulk density (12.61%) compared to ponds subjected to control conditions at the end of the experiment. Over the experiment, improvements in sediment and water quality parameters followed the T2 > T1 > control trend. Growth characteristics showed a significant rise in the percentage of weight gain (12.398 ± 0.12), SGR (11.80 ± 0.01% day-1) and PER (2.39 ± 0.01) with reduced FCR (1.19 ± 0.00) in biochar-treated ponds (T2), followed by T1 and control. The digestive enzyme activity (amylase) and metabolic enzymes like hepatopancreatic alanine aminotransferase (ALT) and muscle ALT activities were significantly higher in shrimps raised in the biochar-applied ponds. On the other hand, the oxidative stress enzyme superoxide dismutase (SOD) in gills, muscles, and hepatopancreas (HP) exhibited lower values, suggesting reduced oxidative stress due to the biochar amendment of the pond sediment. Overall, the study recommends incorporating PSB at a rate of 2 t/ha into nursery pond sediment to enhance water quality, physiological status, and the growth of P. vannamei juveniles. Moreover, this approach improves soil and water characteristics in saline soils, making it an effective culture practice in degraded environments.

Integrated Biomarker Response of Oxidative Stress Parameters in the Digestive Glands and Gills of Autochthonous and Invasive Freshwater Mussels from the Sava River, Serbia.

In this study, the activity of oxidative stress parameters superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST), as well as the concentrations of vitamin E (Vit E) and SH groups in the digestive glands and gills of freshwater mussels Unio pictorum and Sinanodonta woodiana from the Sava River in Serbia were investigated. These parameters were determined in native and invasive mussels under the same environmental conditions. The activities of GSH-Px and GR and the concentration of Vit E were significantly higher in the digestive glands of the autochthonous species U. pictorum than in the invasive species S. woodiana, while the CAT activity and the concentration of SH groups were lower. In the gills of U. pictorum, GSH-Px activity and Vit E concentration were significantly higher, while CAT, GST, and SH groups were lower. Principal component analysis (PCA) showed that oxidative stress parameters were strictly tissue- and species-specific. In addition, integrated biomarker response (IBR) showed a combined response of enzymatic and non-enzymatic oxidative stress parameters depending on the tissue or species studied, indicating different metabolic activities and behaviors of an autochthonous versus an introduced bivalve species.

Salinity influence on Mytilus galloprovincialis exposed to antineoplastic agents: a transcriptomic, biochemical, and histopathological approach

Nowadays, aquatic species face a variety of environmental risks associated with pharmaceutical consumption. More specifically, the increased number of cancer patients has been accompanied by an increased consumption of antineoplastic drugs, such as ifosfamide (IF) and cyclophosphamide (CP). These drugs have been found in aquatic ecosystems, raising concerns about their impact, especially on estuarine species, as marine waters are the final recipients of continental effluents. Simultaneously, predicted climatic changes, such as salinity shifts, may threaten organisms. Considering this, the present research aims to investigate the combined effects of IF and CP, and salinity shifts. For this, a transcriptomic, biochemical, and histopathological assessment was made using the bivalve species Mytilus galloprovincialis exposed for 28 days to IF and CP (500 ng/L), individually, at different salinity levels (20, 30, and 40). IF and CP up-regulated metabolism-related gene cyp3a1, with CP also affecting abcc gene, showing minimal salinity impact and highlighting the importance of these metabolic routes in mussels. Salinity shifts affected the transcription of genes related to apoptosis and cell cycle growth, such as p53, as well as the aerobic metabolism, the antioxidant and biotransformation mechanisms. These findings indicate mussels' high metabolic adaptability to osmotic stress. Under CP exposure and low salinity, mussels exhibited increased cellular damage and histopathological effects in digestive gland tubules, revealing detrimental effects towards M. galloprovincialis, and suggesting that a metabolic slowdown and activation of antioxidant mechanisms helped prevent oxidative damage at the control and high salinities. Overall, results reinforce the need for antineoplastics ecotoxicological risk assessment, especially under foreseen climate change scenarios.

Accumulation, biochemical responses and changes in the redox proteome promoted by Ag and Cd in the burrowing bivalve Scrobicularia plana

Silver (Ag) and cadmium (Cd) are non-essential metals that, as a result of natural processes and human activities, reach the aquatic environment where they interact with biota inducing potential toxic effects. To determine the biological effects of these metals on the endobenthic bivalve Scrobicularia plana, specimens were exposed to Ag and Cd at two concentrations, 5 and 50 μg∙L-1, for 7 days in a controlled microcosm system. The levels of the metals were measured in the seawater, sediments and clam tissues. The possible toxic biological effects of Ag and Cd were studied using a battery of biochemical biomarkers that are responsive to oxidative stress: superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) activities, and metallothioneins (MTs) and lipid peroxidation (LPO) levels. Since both metals have been linked to oxidative stress, redox modifications to proteins were studied by differential isotopic labelling of the oxidised and reduced forms of cysteines (Cys). An accumulation of metals was observed in the digestive gland and gills following exposure, together with the activation of enzyme activities (SOD for the Cd exposure; SOD, CAT, GST, and GR for the Ag exposure). The MT and LPO levels (after individual exposure to Ag and Cd) increased, which suggests the existence of antioxidant and detoxification processes to mitigate the toxic oxidative effects of both metals. The redox proteomic analysis identified 771 Cys-containing peptides (out of 514 proteins), of which 195 and 226 changed after exposure to Ag and Cd, respectively. Bioinformatics analysis showed that exposure to metal affects relevant functional pathways and biological processes in S. plana, such as: “cellular respiration” (Ag), “metabolism of amino acids” and “synthesis and degradation of proteins” (Ag and Cd), “carbohydrate metabolism” and “oxidative stress” (Cd). The proteomic approach implemented here is a powerful complement to conventional biochemical biomarkers, since it evaluates changes at the protein level in a high-throughput unbiased manner, thus providing a general appraisal of the biological responses altered by exposure to the contaminants.

Quantitative tracking of nanoplastics along the food chain from lettuce (Lactuca sativa) to snails (Cantareus aspersus)

Terrestrial systems are a significant sink for plastic contamination, including nano- and microplastics (NMPs). To date, limited information is available about the transfer of NMPs up the food web via trophic transfer, however, concerns about this exposure pathway for invertebrates and higher-level organisms have been raised. We aim to examine and quantify the trophic transfer of europium doped polystyrene nanoplastics (Eu-PS; NPs) within a terrestrial food chain. The uptake of 100 nm spherical Eu-PS particles from water through the roots of the plants to the leaves and finally to garden snails (Cantareus aspersus) was assessed. Lettuce (Lactuca sativa) was cultivated in Hoagland solution spiked with different concentrations of Eu-PS (15, 150 and 1500 μg/L) for three weeks. Then, lettuce shoots were used as food for snails for 19 days at a rate of 1 g of shoots per day. The Eu-PS primarily accumulated in the lettuce roots for all treatments, with a limited transfer to the shoots (only quantifiable in the highest treatment; translocation factor: TF < 1). No detectable levels of Eu-PS were found in the snails' digestive gland; however, the Eu-PS particles were detected in their feces (trophic transfer factor: TFF > 1). Moreover, only limited effects were observed on lettuce biomass by NPs treatments. No effects of the Eu-PS particles on snails were observed, with the exception of a consistent decrease in the shell diameter. Overall, our research illustrates that NPs can be absorbed by plants through their roots, subsequently transported to the shoots. However, our findings show limited transfer of NPs into snail tissues, but direct excretion into their feces. We provide an important insight into the potential transfer within the human food chain.

In vitro and in vivo effects of commercial and environmental microplastics on Unio delicatus.

Microplastics (MPs) are ubiquitous pollutants in freshwater environments. In this study, freshwater mussels, Unio delicatus, were exposed to both environmental MPs (e-MP) and commercial MPs (c-MP) that include green fluorescent MP (gf-MP), polyethylene (c-PE) and polystyrene (c-PS) at environmental concentrations (5 mg/L and 50 mg/L) over duration of 7 and 30 days. According to in vivo experiment results, both e-MPs and c-MPs induced significant changes in the total hemocyte counts of mussels (p < 0.05). Exposure to high concentrations of e-MPs and c-MPs for 7 days led to decreased cellular glutathione levels in the mussels, while exposure to low concentrations of e-MPs and c-PS for 7 days resulted in increased advanced oxidation protein products (AOPP). Mussels exposed to high concentrations of e-MPs for 30 days exhibited decreases in both glutathione levels and AOPP values. Although no damage was observed in tissues other than gills and digestive gland, histopathological alterations were observed in these tissues following exposure to 50 mg/L c-MPs. Additionally, MPs were observed in the intestine tissues. In vitro experiments using the MTT assay showed no significant difference in cell viability between the MP-exposed group and the control group at tested concentrations, with no observed dose-response relationship (p > 0.05). Nevertheless, certain cells exhibited signs of cell death, such as disrupted cellular structures, condensed nuclei, and loss of cellular integrity. These observations were consistent with mechanical compression, indicating that physical contact with MPs may result in cell damage or death. These findings demonstrate that environmentally relevant concentrations of MPs have toxic effects on freshwater mussels and multiple parameters provide valuable insight for the evaluation of health risks of organisms.

Single and combined effects of phenanthrene and cadmium on oxidative stress and detoxification related biomarkers in clams (Meretrix meretrix)

Biomarkers concerning antioxidant reactions and detoxification metabolics were evaluated in Meretrix meretrix exposed to cadmium (Cd, 10 μg/L) and phenanthrene (PHE, 100 μg/L) individually and in combination (10 μg/L Cd + 100 μg/L PHE) for 7 days. The accumulation of Cd and PHE measured in the digestive gland, gill, mantle, and axe foot of the clam showed significant increase in combination treatment and it was higher than the single Cd or single PHE treatment. The activities of oxidative stress-related enzymes, the expression of Cu/Zn SOD, and the content of MDA increased after Cd and PHE exposure in the digestive gland and gill at most cases. In the digestive gland, CAT gene expression was significantly induced in Cd-single group and significantly inhibited in PHE-single group and Cd-PHE mixed group at both day 3 and day 7; in the gill, CAT gene expression was significantly inhibited in all groups at day 3 and except for Cd-single group at day 7. MT expression was significantly induced in Cd-single and Cd-PHE mixed groups at day 7, while hsp70 expression was significantly inhibited in PHE-single and Cd-PHE mixed groups at day 7. The results indicated that SOD, CAT, GST, MDA, Cu/Zn SOD, CAT, MT and hsp70 were sensitive to cadmium and PHE in a water environment, and can be used as indicators of marine heavy metal pollution.

Investigating Non-Native Ribbon Worm Cephalothrix simula as a Potential Source of Tetrodotoxin in British Bivalve Shellfish.

Tetrodotoxin (TTX) is a potent marine neurotoxin found in several phylogenetically diverse organisms, some of which are sought as seafood. Since 2015, TTX has been reported in bivalve shellfish from several estuarine locations along the Mediterranean and European Atlantic coasts, posing an emerging food safety concern. Although reports on spatial and temporal distribution have increased in recent years, processes leading to TTX accumulation in European bivalves are yet to be described. Here, we explored the hypothesis that the ribbon worm species Cephalothrix simula, known to contain high levels of TTX, could play a role in the trophic transfer of the toxin into shellfish. During a field study at a single location in southern England, we confirmed C. simula DNA in seawater adjacent to trestle-farmed Pacific oysters Magallana gigas (formerly Crassostrea gigas) with a history of TTX occurrence. C. simula DNA in seawater was significantly higher in June and July during the active phase of toxin accumulation compared to periods of either no or continually decreasing TTX concentrations in M. gigas. In addition, C. simula DNA was detected in oyster digestive glands collected on 15 June 2021, the day with the highest recorded C. simula DNA abundance in seawater. These findings show evidence of a relationship between C. simula and TTX occurrence, providing support for the hypothesis that bivalves may acquire TTX through filter-feeding on microscopic life forms of C. simula present in the water column at particular periods each year. Although further evidence is needed to confirm such feeding activity, this study significantly contributes to discussions about the biological source of TTX in European bivalve shellfish.

Fate and impact at molecular level of diatrizoic acid and iohexol contrast agents in Dreissena polymorpha mollusks.

Iodinated contrast media (ICMs) used in X-ray imaging for medical diagnostics are released into wastewater and then encountered in river water at concentrations ranging from several dozen to hundreds of µg/L, and even thousands of µg/L in hospital effluents. ICMs are considered as emerging pollutants as their occurrence and impact on ecosystems and the environment are poorly documented. Even if they are considered inert for humans, aquatic organisms are continuously exposed to ICMs, and their potential deleterious effects are therefore questioned as we have recently demonstrated that they enter into organisms such as the zebra mussels. To answer this question, Dreissena polymorpha were exposed to two ICMs of different osmolality, diatrizoic acid and iohexol, at an environmental concentration (100µg/L) for 21days before a depuration phase of 4days. The occurrence, fate, and impact of both ICMs in these organisms were studied using a metallomic approach. Thus, iodine as well as endogenous copper and zinc were quantified and analyzed in cytosolic extracts of digestive glands, gills, and gonads of mussels by size exclusion chromatography coupled to ICP MS. This work shows that a subcellular fractionation is necessary to distinguish variations in total element content. The cytosolic iodoprotein chromatographic pattern was consistent for the three organs and confirmed the presence of ICMs in cytosols. Additionally, this exploratory work tends to show a weak biological effect of ICMs with a substantial variation of the profile of Cu-binding proteins mostly in the gill cytosol and to a lesser extent, in the digestive gland cytosol.

Low-level ionizing radiation-induced DNA responses in the Asian green mussel Perna viridis

Cesium-137 (Cs-137) is a radioactive isotope present in marine environments due to the operation of nuclear power plants and weapons testing. Radiocesium poses a potential risk to marine life due to its long half-life and bioaccumulation. This study evaluated the genotoxicity of low doses of Cs-137 in the Asian green mussel Perna viridis, a sentinel species for marine pollution monitoring, by performing the comet assay and micronucleus test on hemolymph samples. Genotoxicity was assessed after exposing mussels to Cs-137 at dose rates of 0, 5, 10, and 15 μGy/h for 48 h. Cs-137's organ-specific distribution was also determined using HPGe gamma spectrometry. Even at low radiation doses, Cs-137 was found to exert genotoxic effects. Significant increases in DNA strand breaks (%Tail DNA) and micronucleus formation (MNF) were observed at all tested dose rates compared with the levels in controls, with dose-dependent responses. Cs-137 predominantly accumulated in the soft tissues, specifically the gills and digestive gland. The findings support the recommended safety level of 10 μGy/h for aquatic organisms, suggesting its appropriateness as a fundamental criterion for developing the national marine water quality standard for Cs-137 in Thailand.

In vivo assessment of oxidative stress, neurotoxicity and histological alterations induction in the marine gastropod Stramonita haemastoma exposed to Cr2O3 and Al2O3 nanoparticles

The increased use of nanoparticles (NPs) is expected to raise their presence in the marine ecosystem, which is considered as the final destination of released NPs. This study investigated the toxicity of Cr2O3 (42 nm) and Al2O3 (38 nm) NPs (1, 2.5, and 5 mg/L) on the digestive glands of Stramonita haemastoma for 7, 14, and 28 days by oxidative stress biomarkers, neurotoxicity indicator assessment, and histological study. Results revealed an imbalance in antioxidants at all periods. Following 7 days, both NPs caused GSH depletion with marked impacts from Al2O3. GPx, CAT, and AChE were also decreased with the highest changes induced by Cr2O3. Both NPs inducted GSH and GST levels on days 14 and 28, with more effects from Cr2O3 exposure. GPx, AChE, and MDA induction were observed on day 28, while MT varied through NPs and time, with imbalanced levels at all periods noticed, SOD was mostly not affected. Histology revealed alterations including necrosis and interstitial deteriorations; quantitative analysis through the histological condition index revealed dose-dependent impacts, with the highest values attributed to Cr2O3 exposure. While PCA revealed the co-response of GSH, GST, GPx, CAT, and AchE with separated MT responses. This study reported oxidative stress induction through a multi-biomarkers investigation, neurotoxicity, and histological damages in the digestive gland of S. haemastoma following Cr2O3 and Al2O3 NPs exposure.

"Groundbreaking study: Combined effect of marine heatwaves and polyethylene microplastics on Pacific oysters, Crassostrea gigas"

Microplastics (MPs) and rising marine seawater temperatures are one of the major environmental problems threatening the survival of marine organisms and biodiversity. However, interactions between such multiple stressors are virtually unexplored. This study aimed to assess the combined effect of two temperatures and polyethylene MPs on the Pacific oyster Crassostrea gigas, one of the most globalized mollusc species for aquaculture. Our work highlights the potential ecological risk posed by these two factors on marine bivalve molluscs. The experimental design was carried out following a 14-day exposure of oysters to environmental concentrations of polyethylene MPs (0.01 mg.L-1), and to two temperatures (15 °C and 22 °C). Sampling was performed on days 0, 7, and 14. The μ-FTIR analysis was applied to quantify MPs of interest and to check a potential environmental contamination. Tissue samples of digestive glands were collected from the oysters to evaluate the activity of biomarkers including superoxide dismutase, glutathione-s-transferase, malondialdehyde and laccase through protein levels. We note that the combination of MPs and high water temperature (HWT, 22 °C) had a significant impact both on the survival of animals and on stress markers, by modifying lipid peroxidation and immune responses. This original study gave the first innovative results on this topic and provides us with knowledge of the combined effects of MPs pollution and HWT (simulating marine heatwaves situation) on C. gigas. There remains a lack of information on the toxicity and the potential environmental hazard of plastics in the marine environment.

Cyto-architecture of Byblis glands and leaf cells based on freeze-substitution and conventional TEM.

Byblis liniflora (Byblidaceae) is a carnivorous plant that has developed sticky fly paper traps with two types of glandular trichomes producing digestive enzymes and sticky mucilage. This study aimed to analyze the ultrastructure of these glandular leaf trichomes based on rapid freeze fixation and conventional chemical fixation in the attempt to understand their functional contribution to the carnivorous performance of the plants. The Byblis cells were studied in TEM, SEM and STEM using cryo techniques for fixation and substitution in addition to conventional chemical fixation. We show in detail the architecture of both the digestive glands and the mucilage glands with their relevant sets of organelles. Both mitochondria and plastids have a conspicuous plasticity, with branches and constrictions, and they associate to form clusters. The glandular cells appear to be transfer cells with cell wall ingrowths. Digestive glands occur in different states of development. Their cuticle forms discontinuities which are unique among glands of carnivorous plants. They look like cuticular holes -- the cuticle separates from the cell wall in only one spot and then ruptures. Cuticular discontinuities thus differ from cuticular gaps and cuticular pores so far described in carnivorous plants. We therefore propose for them the term cuticular holes. Application of cryo-techniques made it possible to show the true structure of the cell wall and the relationship between cell wall ingrowths and organelles, as well as the morphology and structure of organelles and their associations.

MAPK signaling pathway enhances tolerance of Mytilus galloprovincialis to co-exposure of sulfamethoxazole and polyethylene microplastics

Microplastics (MPs) and antibiotics often coexist in complex marine environments, yet their combined detrimental effects on marine organisms remain underexplored. This study evaluated the effects of polyethylene microplastics (PE, 200 μg/L) and sulfamethoxazole (SMX, 50 μg/L), both individually and in combination, on Mytilus galloprovincialis. The exposure lasted 6 days, followed by a 6-day recovery period. Bioaccumulation, DNA damage, pollutants transport/metabolism related responses and responding alterations of mitogen-activated protein kinase (MAPK) signaling pathway were detected in gills and digestive glands. Bioaccumulation of SMX/PE in mussels occurred in a tissue-specific manner, co-exposure altered SMX contents in investigated tissues. Co-exposure did not induce extra DNA damage, elevated DNA damage was alleviated during the recovery period in all treated groups. The exposure of SMX/PE exerted different alterations in pollutants transport/metabolism related responses, characterized by multixenobiotic resistance and relative expression of key genes (cytochrome P450 monooxygenase, glutathione S-transferase, ATP-binding cassette transporters). Key molecules (p38 MAPK, c-jun N-terminal kinase, extracellular regulated protein kinase, nuclear factor-κB and tumor protein p53) in MAPK signaling pathway were activated at transcriptional and translational levels after SMX/PE and co-exposure. Co-regulation between MAPK members and pollutants transport/metabolism related factors was revealed, suggesting MAPK signaling pathway served as a regulating hub in exposed mussels to conquer SMX/PE stress. Overall, this study provides new insights on SMX/PE induced health risks in marine mussels and potential mechanism through MAPK cascades regulation.

First person – Daisy Pineda-Suazo

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping researchers promote themselves alongside their papers. Daisy Pineda-Suazo is first author on ‘ Evaluation of Octopus maya enzyme activity of the digestive gland and gastric juice’, published in BiO. Daisy is a postdoc in the Applied Ecophysiology laboratory at Universidad Nacional Autónoma de México, Sisal, México, investigating bioinformatics, molecular biology, omics sciences, biochemistry.

Mediterranean mussels (Mytilus galloprovincialis) exposure to fluoxetine: Bioaccumulation and biotransformation products

The significant rise in antidepressant consumption in recent years was accentuated by COVID-19 pandemic. Among these antidepressant, fluoxetine, a selective serotonin re-uptake inhibitor (SSRI), is the most prescribed worldwide. The present study investigated its bioaccumulation and metabolization in the mussel Mytilus galloprovincialis, generally recognized as a reliable bioindicator for assessing environmental quality and the accumulation of various contaminants. Mussels were exposed to a nominal concentration of fluoxetine (3.1 μg/L) for 28 days. Mussels were sacrificed at day 2, 7, 14 and 28 of exposure. The order of accumulation level was gills > digestive glands > soft tissues, and a regular increase in fluoxetine and norfluoxetine was observed across the various sampling days for both digestive glands and soft tissues. The calculated bioconcentration factor (BCF) ranged from 253 at D2 to 1734 at D28 for fluoxetine, and pseudo-BCF from 7 at D2 to 64 at D28 for norfluoxetine. Non-targeted approaches highlighted ten metabolites, which are reported for the first time in Mytilus, in addition to norfluoxetine. Notably, this study highlighted two phase I metabolites and one phase II metabolite previously unreported. These findings contribute to the understanding of fluoxetine accumulation and metabolism in Mytilus and enhance the knowledge of pharmaceuticals detoxification processes in non-target organisms.

Interplay of UV-filter pollution and temperature rise scenarios on Mytilus galloprovincialis health: Unveiling sperm quality and adult physiology, biochemistry, and histology insights

Addressing the impacts of emerging contaminants within the context of climate change is crucial for understanding ecosystem health decline. Among these, the organic UV-filters 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) are widely used in cosmetics and personal care products. Their unique physico-chemical properties, along with their growing commercialization and consumption, have made them ubiquitous in aquatic environments through both direct and indirect releases, raising significant concerns about their potential threats to inhabiting biota. Additionally, increasing surface water temperatures exacerbate ecological risks, making it imperative to understand the implications for non-target species at different biological levels. This study investigated the short- and long-term effects of UV-filters 4-MBC or BP-3, at ecologically relevant concentrations, combined with current and predicted warming scenarios, on the performance and male reproductive health of Mytilus galloprovincialis mussel populations. Using biomarkers across sub-cellular, cellular, tissue, and individual levels, the study revealed significant physiological and biochemical impairments in both sperm cells and adults exposed to UV-filters. Temperature emerged as the primary driver influencing mussel responses and modulating the impacts of 4-MBC/BP-3, emphasizing their sensitivity to temperatures outside the optimal range and interactive effects between stressors. Specifically, sperm motility declined with increasing UV-filter concentrations, while temperature alone influenced ROS production, leading to compromised mitochondrial activity and DNA damage in the presence of combined stressors, indicative of potential reproductive impairments. Adults exhibited high UV-filter bioconcentration potential in whole tissues, compromised physiological status, morphophysiological changes in digestive glands, oxidative stress, and alterations in metabolic capacity, antioxidant defences, and biotransformation mechanisms, correlating with UV-filter exposure and temperature increase. Among the UV-filters tested, 4-MBC was the most detrimental, especially when combined with warming. Overall, this study underscores the vulnerability of M. galloprovincialis to cumulative stressors and highlights the importance of employing a multi-biomarker approach to assess and mitigate the impacts of stressors on coastal ecosystems.

Metabolic variation and oxidative stress responses of clams (Ruditapes philippinarum) perturbed by ofloxacin exposure

Ofloxacin (OFL), one of the most widely used fluoroquinolone antibiotics, has been frequently detected in marine environments. Nonetheless, researchers are yet to focus on the effects of OFL on the benthos. In the present study, marine clams (Ruditapes philippinarum) were exposed to OFL (0.5, 50, and 500 μg/L) for 14 d, followed by a 7 d depuration period. The accumulation of OFL, antioxidative defense responses, neurotoxicity, burrowing behavior, and metabolomic changes in clams were evaluated. The results indicated that OFL could accumulate in clams, albeit with a low bioaccumulation capacity. The intermediate (50 μg/L) and high (500 μg/L) levels of OFL induced significant antioxidative responses in the gills and digestive glands of clams, mainly manifesting as the inhibition of catalase activities and the induction of superoxide dismutase and glutathione S-transferase activities, which ultimately elevated the content of malondialdehyde, causing oxidative damage. Furthermore, the significant induction of acetylcholinesterase activities was observed, coinciding with a significant increase in burrowing rates of clams. The high level of OFL affected glycerophospholipid, arachidonic acid, steroid hormone biosynthesis, unsaturated fatty acids biosynthesis, and glycolysis/glycogenesis metabolism. In conclusion, this study has contributed to the understanding of the physiological and biochemical effects and molecular toxicity mechanisms of OFL to marine bivalves.

Preventing illegal seafood trade using machine-learning assisted microbiome analysis

BackgroundSeafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems).ResultsMachine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score.ConclusionsWe show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish.