Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Integrating pollutant levels and biochemical biomarkers in oysters (Crassostrea rhizophorae and Crassostrea gasar) indicates anthropic impacts on marine environments along the coast of Santa Catarina state, Brazil.

This study aimed to carry out a general diagnosis of the contamination of the coastal marine environment of the Santa Catarina state (SC, Brazil) by different classes of environmental pollutants, as well as to evaluate possible adverse effects of the contaminants on biochemical biomarkers of oysters, Crassostrea gasar and Crassostrea rhizophorae. 107 chemicals were evaluated in water, sediment and oyster samples from nine sites along the coastline of SC. We also examined various biochemical biomarkers in the oysters' gills and digestive glands to assess potential effects of contaminants. In general, the northern and central regions of the littoral of SC presented higher occurrences and magnitudes of contaminants than the southern region, which is probably related to higher urbanization of center and northern areas of the littoral. The biomarker analysis in the oysters reflected these contamination patterns, with more significant alterations observed in regions with higher levels of pollutants. Our results may serve as a first baseline for future and more extensive monitoring actions and follow-up of the degree of contamination in the state, allowing for inspection actions and management of areas most affected by marine pollutants.

TRIM21 chimeric protein as a new molecular tool for multispecies IgG detection.

BACKGROUND: The production of monoclonal antibodies for immunoglobulin detection is not cost-effective, while polyclonal antibody production depends on laboratory animals, raising concerns on animal welfare. The widespread use of immunoglobulins in the pharmaceutical industry and the increasing number and variety of new antibodies entering the market require new detection and purification strategies. The Tripartite motif-containing protein 21 is a soluble intracellular immunoglobulin G receptor that binds to the constant region of immunoglobulin G from various species with high affinity. We hypothesized that using this protein as an antibody-binding module to create immunoglobulin detection probes will improve the portfolio of antibody affinity ligands for diagnostic or therapeutic purposes. RESULTS: We created a chimeric protein containing a mutated form of the C-terminal domain of mouse Tripartite motif-containing protein 21 linked to streptavidin to detect immunoglobulin G from various species of mammals. The protein is produced by heterologous expression and consists of an improved molecular tool, expanding the portfolio of antibody-affinity ligands for immunoassays. We also demonstrate that this affinity ligand may be used for purification purposes since imidazole elution of antibodies can be achieved instead of acidic elution conditions of current antibody purification methods. CONCLUSION: Data reported here provides an additional and superior alternative to the use of secondary antibodies, expanding the portfolio of antibodies affinity ligands for detection and purification purposes.

Biochemical biomarkers and metals in Perna perna mussels from mariculture zones of Santa Catarina, Brazil.

The activity of cholinesterase (ChE), glutathione-S transferase (GST), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH) and catalase (CAT) was evaluated in the gill and digestive glands of the Perna perna mussel transplanted to three non-contaminated mariculture zones under the influence of distinct physical-chemical characteristics. Differences among sites for ChE, GST and CAT activities in gill, as well as ChE, GST and G6PDH activity in digestive gland of mussels, were found and possibly related to differences in physicochemical characteristics of the sites and/or biological status of the mussels. Mussels that were transplanted to another, more urbanized site (Ponta do Lessa) with similar physicochemical characteristics to one of the farming sites (Sambaqui), was also chosen to evaluate biomarker responses to pollution. Activities of ChE, GST and GR in the digestive glands and CAT in the gills were higher in the polluted site. GR was the only biomarker to be unaltered in different farming sites, but induced in the pollution site. The trace metal concentrations in the mussels were low and unlikely to cause the changes observed in the biomarker levels. The present study strongly suggests that monitoring programs should compare sites with similar physicochemical characteristics when using a complementary biomarker approach. In addition, the baselines for the biomarkers and metal used in the present study can serve as a reference for the monitoring of these mariculture zones in future monitoring programs employing P. perna.

Differential gene expression in oyster exposed to sewage.

In order to investigate the influence of domestic sewage on the gene expression of Pacific oysters Crassostrea gigas, suppression subtractive hybridization (SSH) method was employed. Oysters were sampled at a farming area and, after 10 days of acclimation in the laboratory, were exposed to untreated domestic sewage diluted 33% for 48 h. Gills of male oysters were excised for total RNA extraction. mRNA was purified and the differential gene expression was analyzed by SSH. We obtained 61 cDNA sequences but only 15 were identified, which includes fatty acid binding protein, multidrug resistance protein, omega glutathione S-transferase, cytochrome P450 isoform CYP356A1, among others. The identified genes are associated with different metabolic functions like biotransformation, membrane transport, aerobic metabolism and translational machinery, evidencing the potential toxic effects elicited by these effluents.

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, ß-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.

Identification of differentially transcribed genes in shrimp Litopenaeus vannamei exposed to osmotic stress and challenged with WSSV virus.

The effects of hyposmotic stress and white spot syndrome virus (WSSV) challenge in expression was studied in the marine shrimp Litopenaeus vannamei. Messenger RNA from gills of shrimp submitted to osmotic stress was isolated to identify genes differentially expressed through the suppressive subtractive hybridization (SSH) method. Two subtractive libraries forward and two reverse were constructed to identify up and down-regulated genes under these conditions. About 192 clones were sequenced, of which 46 genes were identified. These genes encode proteins corresponding to a wide range of biological roles, including defense, cell signaling, electron transfer, cell proliferation and differentiation, apoptosis, intermediary metabolism, cytoskeleton and digestion. Among the identified genes, 19 were up-regulated and 27 were down-regulated in the animals kept at a lower ion concentration. We evaluated the expression of eight genes by RT-qPCR in shrimp submitted to hyposmotic conditions with and without WSSV challenge. The SSH enabled the identification of genes that are influenced by hyposmotic stress. A significant up-regulation was observed in lectin-C, QM, TGF beta inducible nuclear protein 1, ciclophilin, malate dehydrogenase, mitochondrial ATP synthase F chain and ATP synthase subunit 9 precursor transcripts. However, the expression of these genes in L. vannamei was not affected by WSSV infection both at isosmotic and hyposmotic conditions.

Salinity influences glutathione S-transferase activity and lipid peroxidation responses in the Crassostrea gigas oyster exposed to diesel oil.

Biochemical responses in bivalve mollusks are commonly employed in environmental studies as biomarkers of aquatic contamination. The present study evaluated the possible influence of salinity (35, 25, 15 and 9ppt) in the biomarker responses of Crassostrea gigas oysters exposed to diesel at different nominal concentrations (0.01, 0.1 and 1mL.L(-1)) using a semi-static exposure system. Salinity alone did not resulted in major changes in the gill's catalase activity (CAT), glutathione S-transferase activity (GST) and lipid peroxidation levels (measured as malondialdehyde, MDA), but influenced diesel related responses. At 25ppt salinity, but not at the other salinity levels, oysters exposed to diesel showed a strikingly positive concentration-dependent GST response. At 25ppt and 1mL.L(-1) diesel, the GST activity in the gills remained elevated, even after one week of depuration in clean water. The increased MDA levels in the oysters exposed to diesel comparing to control groups at 9, 15 and 35ppt salinities suggest the occurrence of lipid peroxidation in those salinities, but not at 25ppt salinity. The MDA quickly returned to basal levels after 24h of depuration. CAT activity was unaltered by the treatments employed. High toxicity for 1mL.L(-1) diesel was observed only at 35ppt salinity, but not in the other salinities. Results from this study strongly suggest that salinity influences the diesel related biomarker responses and toxicity in C. gigas, and that some of those responses remain altered even after depuration.

Effects of salinity on biomarker responses in Crassostrea rhizophorae (Mollusca, Bivalvia) exposed to diesel oil.

Crassostrea rhizophorae is a euryhaline oyster that inhabits mangrove areas, which are widely distributed along the Brazilian coast. The aim of this study was to investigate the effects of salinity (9, 15, 25, and 35ppt) on the activities of glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), catalase (CAT), and acetylcholinesterase (AChE) in the digestive gland of this species after exposure to diesel oil for 7 days at nominal concentrations of 0.01, 0.1, and 1mlL(-1) and after depuration for 24h and 7 days. GST activity increased in a diesel oil concentration-dependent manner at salinities 25 and 15ppt and remained slightly elevated even after depuration periods of 24h and 7 days. No changes were observed in the activities of G6PDH, CAT, and AChE in the oysters exposed to diesel and depurated. Based on these results, GST activity in the digestive gland of C. rhizophorae might be used as a biomarker of exposure to diesel oil in sites where the salinity is between 15 and 25ppt, values usually observed in mangrove ecosystems.