Transcriptomic investigation and biomarker discovery for zinc response in oysters Crassostrea gasar.

In an era of unprecedented industrial and agricultural growth, metal contamination in marine environments is a pressing concern. Sentinel organisms such as the mangrove oyster Crassostrea gasar provide valuable insights into these environments' health. However, a comprehensive understanding of the molecular mechanisms underlying their response to metal exposure remains elusive. To address this gap, we reanalyzed the 454-sequencing data of C. gasar, utilizing an array of bioinformatics workflow of CDTA (Combined De Novo Transcriptome Assembly) to generate a more representative assembly. In parallel, C. gasar individuals were exposed to two concentrations of zinc (850 and 4500 µg L-1 Zn) for 48 h to understand their molecular responses. We utilized Trinotate workflow for the 11,684-CDTA unigenes annotation, with most transcripts aligning with the genus Crassostrea. Our analysis indicated that 67.3% of transcript sequences showed homology with Pfam, while 51.4% and 54.5%, respectively had GO and KO terms annotated. We identified potential metal pollution biomarkers, focusing on metal-related genes, such as those related to the GSH biosynthesis (CHAC1 and GCLC-like), to zinc transporters (ZNT2-like), and metallothionein (MT-like). The evolutionary conservation of these genes within the Crassostrea genus was assessed through phylogenetic analysis. Further, these genes were evaluated by qPCR in the laboratory exposed oysters. All target genes exhibited significant upregulation upon exposure to Zn at both 850 and 4500 µg L-1, except for GCLC-like, which showed upregulation only at the higher concentration of 4500 µg L-1. This result suggests distinct activation thresholds and complex interactions among these genes in response to varying Zn concentrations. Our study provides insights into the molecular responses of C. gasar to Zn, adding valuable tools for monitoring metal pollution in marine ecosystems using the mangrove oyster as a sentinel organism.

Emerging and legacy contaminants on the Brazilian southern coast (Santa Catarina): A multi-biomarker approach in oysters Crassostrea gasar (Adanson, 1757).

Coastal environments, such as those in the Santa Catarina State (SC, Brazil), are considered the primary receptors of anthropogenic pollutants. In this study, our objective was to evaluate the levels of emerging contaminants (ECs) and persistent organic pollutants (POPs) in indigenous Crassostrea gasar oysters from different regions of SC coast in the summer season (March 2022). Field collections were conducted in the São Francisco do Sul, Itajaí, Florianópolis and Laguna coastal zones. We analyzed the bioaccumulation levels of 75 compounds, including antibiotics (AB), endocrine disruptors (ED), non-steroidal anti-inflammatory drugs (NSAIDs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Furthermore, we assessed biomarker responses related to biotransformation, antioxidant defense, heat shock protection and oxidative damage in oysters' gills. Prevalence of ECs was observed in the central and southern regions, while the highest concentrations of POPs were detected in the central-northern regions of SC. Oysters exhibited an induction in biotransformation systems (cyp2au1 and cyp356a1, sult and GST activity) and antioxidant enzymes activities (SOD, CAT and GPx). Higher susceptibility to lipid peroxidation was observed in the animals from Florianópolis compared to other regions. Correlation analyses indicated possible associations between contaminants and environmental variables in the biomarker responses, serving as a warning related to climate change. Our results highlight the influence of anthropogenic activities on SC, serving as baseline of ECs and POPs levels in the coastal areas of Santa Catarina, indicating more critical zones for extensive monitoring, aiming to conserve coastal regions.

Biochemical responses of the mussel Perna perna after exposure to environmental concentrations of the UV filter Benzophenone-3.

Personal care products, such as UV filters, are frequently present in aquatic ecosystems, but studies on their impact on marine organisms are still scarce. Here we addressed the effects of benzophenone-3 (BP-3) on the antioxidant status of Perna perna mussels exposed to concentrations of 0.1 and 3 µg.L-1 for 72 h and 7 days. Glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) activity and lipoperoxidation (MDA) were evaluated in the gills. A significant reduction (p < 0.05) in the activity of G6PDH and GPx was observed after exposure for 7 days to 0.1 µg.L-1. However, no significant differences were observed in GST activity and MDA levels, independently of the exposure time. Principal component analysis (PCA) showed an association of BP-3 highest concentration with GR and MDA at 72 h and only with GR at 7 days of exposure. Similarly, the integrated biomarker response (IBR) demonstrated GR and MDA alterations. In conclusion, environmentally relevant concentrations of BP-3 altered antioxidant and auxiliary enzymes, which could cause long-term damage to P.perna mussels. The need to implement more efficient techniques in wastewater treatment systems is pointed out, especially in summer, when UV filters are used more frequently and abundantly.

Comparative biochemical and molecular responses of biotransformation and antioxidant systems in three species of Crassostrea (Sacco, 1897) oysters exposed to chrysene.

Chrysene (CHR) is among the most persistent polycyclic aromatic hydrocarbons (PAH) in water and a priority compound for pollutants monitoring, due to its carcinogenic, mutagenic and genotoxic potential. Aquatic animals exposed to CHR may present alterations of biomarkers involved in the biotransformation and oxidative stress-related parameters. The aim of this study was to investigate differences in antioxidant and biotransformation (phase I and II) systems of Crassostrea gigas, C. gasar and C. rhizophorae and its effects resulting from CHR exposure. Adult oysters of these species were exposed to 10 µg L-1 of CHR for 24 h and 96 h. In gills, the transcripts CYP1-like, CYP2-like, CYP2AU1-like, GSTO-like, MGST-like, SULT-like were evaluated after 24 h of exposure. The activity of SOD, CAT, GPx, GR and G6PDH were analyzed in gills and digestive glands after 96 h of exposure. CHR bioaccumulated in tissues. Differences in the remaining levels of CHR in water after 96 h were observed in aquaria containing C. gigas or C. gasar oysters and may be associated to the different filtration rates between these species. Downregulate of biotransformation genes were observed in gills of C. gasar (CYP2AU1-like and GSTO-like) and C. rhizophorae (CYP1-like1, CYP2-like, MGST-like and SULT-like), suggesting that biotransformation responses may be species-specific. Differential activity of antioxidant enzymes were observed in gills and digestive gland of oysters exposed to CHR. Biochemical responses suggested that C. gigas and C. gasar are more responsive to CHR. Differential responses observed among the three Crassostrea species can be related to evolutionary differences, ecological niches and adaptation to environment.

Microplastics and linear alkylbenzene levels in oysters Crassostrea gigas driven by sewage contamination at an important aquaculture area of Brazil.

The levels of linear alkylbenzenes (LABs) and the occurrence of microplastics (MPs) in the oysters Crassostrea gigas were evaluated in six farming areas in southern Brazil. The results revealed higher concentrations of LABs in oyster tissue from the Serraria (1977 ± 497.7 ng g-1) and Imaruim (1038 ± 409.9 ng g-1) sites. Plastic microfibers were found in oysters from all locations with values from 0.33 to 0.75 MPs per oyster (0.27-0.64 MPs per gram) showing the ubiquitous presence of this contaminant in the marine environment, which could be considered a threat to farming organisms. In addition, elements such as Ti, Al, Ba, V, Rb, Cr, and Cu were found in the chemical composition of the microfibers, suggesting MPs as vectors of inorganic compounds. A positive correlation between LABs and thermotolerant coliforms suggests that sewage discharges are the main source of contamination in these oysters cultured for human consumption. The present study highlights the need for efficient wastewater treatment plants and the implementation of depuration techniques in oysters from farming areas.

Molecular changes in oysters Crassostrea gigas (Thunberg, 1793) from aquaculture areas of Santa Catarina Island bays (Florianópolis, Brazil) reveal anthropogenic effects.

Anthropogenic activities in coastal regions cause risks to the environmental and human health. Due to the carcinogenic and mutagenic potential, polycyclic aromatic hydrocarbons (PAH) are considered priority for monitoring. Most of the Brazilian production of Crassostrea gigas oysters are placed in the Bays of Santa Catarina Island. The aim of this study was to evaluate molecular responses (phase I and II of biotransformation and antioxidant defense) of C. gigas from six oyster farming areas potentially contaminated by sanitary sewage in Florianópolis Metropolitan (SC, Brazil): Santo Antônio de Lisboa, Sambaqui, Serraria, Caieira, Tapera, Imaruim. We evaluated the transcript levels of CYP1A1-like, CYP2-like, CYP2AU2-like, CYP356A1, GSTA1A-like, GSTO.4A-like, SULT-like, SOD-like and CAT-like by qRT-PCR. Only oysters from Caieira showed levels of thermotolerant coliforms allowed by the law. Chemicals analyses in soft tissues of oysters showed low to average levels of PAH in all monitored areas. Enhanced transcript levels of phase I (CYP1A1-like, CYP3564A1-like, CYP2-like and CYP2AU2-like) were observed in oysters from Serraria and Imaruí, suggesting higher biotransformation activity in these farming areas. Regarding phase II of biotransformation, GSTO.4A-like was up-regulated in oysters from Imaruí compared to Caieira and Santo Antônio de Lisboa. An upregulation of SOD-like and CAT-like were observed in oysters from Imaruí and Serraria, suggesting that oysters from these sites are facing higher prooxidant conditions compared to other areas. By integrating the biological and chemical data it is suggested that human-derived contaminants are affecting the oyster metabolism in some farming areas.

Metal bioaccumulation, oxidative stress and antioxidant responses in oysters Crassostrea gasar transplanted to an estuary in southern Brazil.

The present study assessed the spatial and temporal variations on metal bioaccumulation and biochemical biomarker responses in oysters Crassostrea gasar transplanted to two different sites (S1 and S2) at the Laguna Estuarine System (LES), southern Brazil, over a 45-days period. A multi-biomarker approach was used, including the evaluation of lipid peroxidation (MDA) levels, and antioxidant defense enzymes (CAT, GPx, GR and G6PDH) and phase II biotransformation enzyme (GST) in the gills and digestive gland of oysters in combination with the quantification of Al, Cd, Cu, Pb, Fe, Ni and Zn in both tissues. The exposed oysters bioaccumulated metals, especially Al, Cd and Zn in gills and digestive gland, with most prominent biomarker responses in the gills. Results showed that GPx, GR and G6PDH enzymes offered an increased and coordinated response possibly against metal (Zn, Ni, Cd and Cu) contamination in gills. GST was inversely correlated to Cd levels, being its activity significantly lowered over the 45-d exposure periods at S2. On contrary, in digestive gland GST was slightly positively correlated to Cd, revealing a compensatory mechanism between tissues to protect oysters' cells against oxidative damages, since MDA levels also decreased. CAT also appeared to be involved in the cellular protection against oxidative stress, being increased in gills. However, CAT was negatively correlated to Al levels, which might suggest a possible inhibitory effect of this metal in the gills of C. gasar. Differences between tissues were evident by the Integrative Biomarker Responses version 2 (IBRv2) indexes, which showed different pattern between tissues when studying the sites and exposure periods separately. This study provided evidence for the effectiveness of using a multi-biomarker approach in oyster C. gasar to monitor estuarine metal pollution.

Exposure to phenanthrene and depuration: Changes on gene transcription, enzymatic activity and lipid peroxidation in gill of scallops Nodipecten nodosus.

Understanding the mechanism of phenanthrene (PHE) biotransformation and related cellular responses in bivalves can be an important tool to elucidate the risks of polycyclic aromatic hydrocarbons (PAHs) to aquatic organisms. In the present study it was analyzed the transcriptional levels of 13 biotransformation genes related to cytochrome P450 (CYP), glutathione S-transferase (GST), sulfotransferase (SULT), flavin-containing monooxygenase and fatty acid-binding proteins by qPCR in gill of scallops Nodipecten nodosus exposed for 24 or 96h to 50 or 200µgL(-1) PHE (equivalent to 0.28 and 1.12µM, respectively), followed by depuration in clean water for 96h (DEP). Likewise, it was quantified the activity of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), GST and levels of lipid peroxidation. Increased transcriptional levels of CYP2UI-like, CYP2D20-like, CYP3A11-like, GSTomega-like, SULT1B1-like genes were detected in organisms exposed to PHE for 24 or 96h. In parallel, GR and GPX activities increased after 96h exposure to 200µgL(-1) PHE and G6PDH activity increased after 24h exposure to 50µgL(-1) PHE. This enhancement of antioxidant and phase I and II biotransformation systems may be related to the 2.7 and 12.5 fold increases in PHE bioaccumulation after 96h exposure to 50 and 200µgL(-1) PHE, respectively. Interestingly, DEP caused reestablishment of GPX and GR activity, as well as to the transcript levels of all upregulated biotransformation genes (except for SULT1B1-like). Bioaccumulated PHE levels decreased 2.5-2.9 fold after depuration, although some biochemical and molecular modifications were still present. Lipid peroxidation levels remained lower in animals exposed to 200µgL(-1) PHE for 24h and DEP. These data indicate that N. nodosus is able to induce an antioxidant and biotransformation-related response to PHE exposure, counteracting its toxicity, and DEP can be an effective protocol for bivalve depuration after PHE exposure.

Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen.

Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 µg L(-1). Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-π-like), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock protein-like (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 µg L(-1). A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COX-like, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 µg L(-1) for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 µg L(-1)) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.