Validation of reference genes for RT-qPCR in marine bivalve ecotoxicology: Systematic review and case study using copper treated primary Ruditapes philippinarum hemocytes

The appropriate selection of reference genes for the normalization of non-biological variance in reverse transcription real-time quantitative PCR (RT-qPCR) is essential for the accurate interpretation of the collected data. The use of multiple validated reference genes has been shown to substantially increase the robustness of the normalization. It is therefore considered good practice to validate putative genes under specific conditions, determine the optimal number of genes to be employed, and report the method or methods used. Under this premise, we assessed the current state of reference gene based normalization in RT-qPCR bivalve ecotoxicology studies (post 2011), employing a systematic quantitative literature review. A total of 52 papers met our criteria and were analysed for genes used, the use of multiple reference genes, as well as the validation method employed. We further critically discuss methods for reference gene validation based on a case study using copper exposed primary hemocytes from the marine bivalve Ruditapes philippinarum; including the established algorithms geNorm, NormFinder and BestKeeper, as well as the popular online tool RefFinder. We identified that RT-qPCR normalization is largely performed using single reference genes, while less than 40% of the studies attempted to experimentally validate the expression stability of the genes used. 18s rRNA and β-Actin were the most popular genes, yet their un-validated use did introduce artefactual variance that altered the interpretation of the resulting data. Our findings further suggest that combining the results from multiple individual algorithms and calculating the overall best-ranked gene, as computed by the RefFinder tool, does not by default lead to the identification of the most suitable reference genes.This work was funded by the regional government of Andalusia (Junta de Andalucía) project PE2011-RNM-7812, as well as supported by the Erasmus Mundus Ph.D. fellowship in Marine and Coastal Management to M. V. (as coordinated by the University of Cadiz, Spain). M.H. is supported by a Ramón y Cajal contract (RYC-2012-12217) from the Spanish Ministry of Economy and Competitiveness (MINECO). The authors would further like to express their gratitude to Dr. Cajaraville and Dr. Katsumiti from the Department of Zoology and Animal Cell Biology at the University of the Basque Country (UPV/EHU) for their help in establishing the primary hemocyte cultures.Peer reviewe

Itraq-based proteomics analysis of citrate gold nanoparticle exposure effects in the marine clam Ruditapes phlippinarum

Trabajo presentado en el 10th Iberian Congress on Environmental Contamination and Toxicology y el 7th Iberoamerican Congress on Environmental Contamination and Toxicology (CICTA 2015), celebrados en Vila Real (Portugal) del 14 al 17 de julio de 2015.Engineered Gold nanoparticles (AuNPs) are introduced into a growing number of commercial products. Notwithstanding their promising applications, the increased likelihood of their release into environmental compartments and the resulting exposure on the species within needs to be met by an increased understanding of their behaviour and effects. Up to date limited information is available on the ecotoxicological risk for non-target organisms, with the majority of research focusing on magnified expectable environmental concentrations. In our laboratory-based study the bivalve Ruditapes philippinarum was chosen as a model to evaluate differential protein expression of citrate capped AuNPs (21.5 ± 2.9 nm) at an environmental relevant concentration (0.75 μg L-1). We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify and quantify differently expressed proteins in the digestive gland after 1 and 7 days of exposure in comparison to control organisms.The iTRAQ results demonstrate that the digestive gland is affected by environmentally relevant concentrations of citrate capped gold nanoparticles. Over 2500 proteins involved in a wide range of metabolic pathways and/or physiological processes were identified. Altered proteins may be useful as biomarkers of environmental nanoparticle pollution and provide insight about the mode of action of engineered gold nanoparticles.N

Assessing lead toxicity in the clam Ruditapes philippinarum: Bioaccumulation and biochemical responses

Lead (Pb) is a non-essential metal. Its occurrence in the environment is related principally to anthropogenic contamination. Pb is toxic to aquatic organisms and can provoke damage to membranes and inhibit the activity of essential enzymes. The filter-feeding, Manila clam Ruditapes philippinarum is widely used as a biomonitor organism to assess metal toxicity. Among biomarkers related to the Pb toxicity, the enzymatic activity of δ-aminolevulinic acid dehydratase (δ-ALAD) has been adopted as a specific tool. Metallothionein (MT), lipid peroxidation (LPO) and antioxidant enzymes activities, such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) and superoxidase dismutase (SOD) have also been employed to assess the toxic effect of metals. Two target tissues, the gills and the digestive gland, were selected to examine biomarker responses. In order to assess the effects of Pb accumulation and the mechanisms involved in the recovery from it, clams were exposed at two Pb levels (10 and 100 µg/L) for 7 days and were later maintained in clean water for 7 days as a depuration period. Pb accumulation was dependent on the exposure concentration and higher Pb levels were observed in the gills compared to the digestive gland. Inhibition of δ-ALAD, GST and SOD and the induction of MT and LPO over the exposure period were observed in the gills and the digestive gland of R. philippinarum. The depuration period showed a continuous inhibition of the δ-ALAD activity and induction of MT and LPO in both tissues. These results demonstrate that lead induced an exposure effect and the 7 days of depuration were not sufficient to recover the basal health status of the clams.Peer reviewe

Henri Michaud. Jésus selon le Coran

Rodinson Maxime. Henri Michaud. Jésus selon le Coran. In: Revue de l'histoire des religions, tome 163, n°1, 1963. pp. 97-99

I-TRAQ proteomic approach to understand low concentration citrate capped gold nanoparticle stress in the marine bivalve Ruditapes philippinarum

Trabajo presentado en el 6th Congress of the Spanish Proteomic Society, celebrado en Cádiz del 15 al 18 de noviembre de 2016.Over the last decades engineered gold nanoparticles (AuNPs) have been developed for and introduced into a growing number of commercial and industrial products. Up to date limited information is available on the ecotoxicological responses in non-target organisms, in par-ticular under environmental relevant concentrations. Notwithstanding their promising applica-tions, increased understanding of their behaviour and effects is required to manage potential risk for non-target organisms.We studied differential protein expression in response to citrate capped AuNPs (21.5 ± 2.9 nm) in the digestive gland of the bivalve Ruditapes philippinarum after 1 and 7 days of expo-sure at an environmental relevant concentration (750 ng L-1), using a 2nd-generation (iTRAQ-8plex) proteomic approach.2200 proteins were identified to be simultaneously expressed in control and treatment and a regularized regression approach (Elastic Net) identified 105 of those to be expressed dif-ferentially. Homology-based functional annotation could be successfully performed for 77% of all sequences and 75% of the differentially expressed sequences and subsequent analysis identified potential enrichment in various stress and injury related functions. Identified pro-teins may be useful as biomarkers of environmental nanoparticle pollution and provide insight about the mode of action of engineered gold nanoparticles.N

Assesing toxicity of citrate-gold nanoparticles at different marine trophic levels (microalgae, copepods and bivalve mollusks)

Ponencia presentada en la NanoSpain Conference, celebrada en Santander del 27 de febrero al 1 de marzo de 2012.Engineered nanoparticles (ENPs) may offer benefits to society in general, although they sometimes inherently have unintended effects on ecosystems. As a consequence, assessment of the environmental safety of ENPs has become a major issue worldwide [1]. Within the metallic ENPs, gold nanoparticles have been used extensively in drug delivery, gene therapy, biosensing and contrast agent for imaging [2]. However, studies about the effects of gold nanoparticles are limited and they are specially focused on ¿in vitro¿ experiments rather than ¿in vivo¿ systems. Additionally, estuaries and coastal ecosystems are the final receptors of substances dumped in the environment wherefore the effects of these substances should be tested in representative site specific organisms. In order to assess the effect of gold-citrate nanoparticles on aquatic ecosystems, toxicity tests were carried out in three groups of model organisms belonging to different trophic levels: the marine microalgae species Cylindrotheca closterium, Chlorella autotrophyca, Phaeodactylum tricornutum, Pleurochrysis pseudoroscoffensis and Rhodomonas salina (Fig. 1), the copepod, Tisbe battagliai (Fig. 2) and the clam Ruditapes philippinarum (Fig. 3). The gold-citrate NPs employed were citrate reduced AuNPs in the range of 20 ¿ 30 nm, or soluble gold, H(AuCl4) as positive control. For the toxicity test with microalgae, the selected endpoint was population growth after 72 hours of exposure. The cells were incubated in batch cultures of 50 mL in artificial seawater enriched with simple medium (nitrate, phosphate, silica) and exposed under continuous light conditions at 20±1ºC to different dissolved Au or NPs concentrations. Growths of experimental populations were compared with controls, and concentrations which imply an inhibition of 50% respect the controls (EC50%) are calculated (Fig. 4). Dissolved Au toxicity ranged from 0.052 ± 0.001 mg¿L-1 for Rhodomonas salina to 0.50 ± 0.15 mg¿L-1 for Chlorella autotrophyca. Concentrations at ecologically significant values for NPs (up to 0.3 mg¿L-1) did not imply growth inhibitions over 50%. For copepods, nauplii (< 24 h-old) were exposed (48 h) to increasing concentrations of Au-NPs in 12-well plates (5 ml/well, 4 nauplii/well and 5 replicates/concentration) under the above described laboratory conditions [3]. The results are shown in Figure 5. The clam, Ruditapes philippinarum was exposed for 28 days to two Au-NPs concentrations: 6 and 30 ¿g·L-1. Clams were collected different at sampling points and target tissues (gills, digestive gland and mantel) were dissected and stored at -80ºC until their analysis. No significant mortality was recorded during the experiment and bioaccumulation in the digestive gland along the experiment was measured (Figure 6). In summary, no acute toxicity was recorded at ecological relevant concentrations for assayed Au-NPs. Nevertheless, further research should be necessary to know the effect of chronic exposure to these NPs and to improve the knowledge about their environmental risk assessment.Peer Reviewe

Biomolecular coronas in invertebrate species: Implications in the environmental impact of nanoparticles

The development of nanotechnology will inevitably lead to the release of consistent amounts of nanoparticles (NPs) in the environment. Invertebrates, that represent &gt; 90% of animal species, widespread in different ecosystems, are emerging both as suitable target organisms and as models for evaluating the environmental impact of NPs. Once released in different ecosystems, both NP intrinsic properties and those of the receiving medium will affect particle behavior. In particular, interactions with different biomolecules will lead to the formation of \ue2\u80\u98eco-coronas\ue2\u80\u99, that will influence NP bioavailability/uptake/toxicity in different environments, depending on the organisms present and the surrounding conditions. However, as shown in mammalian studies, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment, i.e. in biological fluids. Different types of NPs associate with serum soluble components, organized into a \ue2\u80\u98protein corona\ue2\u80\u99, which confers a biological identity to NPs and affects their interactions with target cells. In comparison, the study on NP-protein coronas in invertebrates is still at its infancy, since the protein composition of their extracellular fluids is largely unknown, given the large diversity of phyla and species. From the first demonstration of an AgNP protein corona in the body fluid of earthworms, NP-protein coronas are being characterized in marine invertebrates. The identification of protein coronas formed with different types of NPs (amino modified polystyrene- PS NH2 and nano-oxides, n-CeO2 n-TiO2) in hemolymph serum of the marine bivalve Mytilus galloprovincialis is presented as a case study. The results indicate that, in Mytilus hemolymph, the formation of a biomolecular corona is partly NP-specific. The results obtained so far in terrestrial and marine invertebrates indicate that in each model, endowed with a peculiar protein repertoire, NP-coronas are characterized by unique protein components. The role of NP-protein coronas formed by lower organisms, and their possible contribution in evaluating the environmental impact of NPs are discussed

Uptake, elimination and oxidative stress response to 0.75 ug/L citrate gold nanoparticle exposure in the marine clam E. philippinarum

Trabajo presentado en el 2nd Marine NanoEcoSafety Workshop (MANET), celebrado en Palermo el 17 y 18 de noviembre de 2014.Gold nanopar ticles (AuNPs) h ave unique physical and chemical properties and are therefore used and developed for a wide r ange of novel applications in medicine, biology and chem istry. However in recent years concern has been raised over their ability to enter cells, organelles and n uclei, and provoke oxidative stress. In a laboratory-based experiment the non -t arget marine bivalve Ruditapes philippinarum was used as a model organismo Uptake, elimination and molec ular effects of ~20 nm citrate AuNPs at 0.75 Ilg 1-1 were studied over 7 days exposure and equal length elimination periodo Our results demonstrate that the AuNPs were readily taken up into the digestive glan d > gilIs. We further observed limited oxidative stress and inflammatory responses. The activity of several antioxidant en zy mes was increased in an organ sp ecific manner, with the digestive gland ex.hibiting the majority of its enzyme activity within 2,1 hours of exposure. Gene expression analysis highlighted the involvement of inflammatory re sponse genes in digestive gland tissue, as measured by changes in T NF-a and Gadd45 expression. Suggesting that one m echanism of citrate AuNP toxicity i5 well described by the oxidative stress par adigm. Further a significant elimination of Au from the digestive tract within t he purification period was observed, with excretion being an important pathway. In conclusiol1, wh ile not producing significant oxidative dam ag e, concern is rajsed regardi ng the effects of ch ronic AuNP exposure.N

Citrate gold nanoparticle exposure in the marine bivalve Ruditapes philippinarum: uptake, elimination and oxidative stress response

Gold nanoparticles (AuNPs) are considered an important nano-sized component of the twenty-first century. Due to their unique physical and chemical properties, they are being used and developed for a wide range of promising applications in medicine, biology and chemistry. Notwithstanding their useful aspects, in recent years concern has been raised over their ability to enter cells, organelles and nuclei and provoke oxidative stress. In a laboratory-based experiment, the non-target marine bivalve Ruditapes philippinarum was used as a model organism. Uptake, elimination and molecular effects under short-term and sub-chronic exposure conditions to an environmental relevant concentration (0.75 μg L−1) of weakly agglomerating citrate AuNPs (∼20 nm) were studied. Our results demonstrate that at the tested concentration, the particles are readily taken up into the digestive gland > gills and can produce significant changes (p < 0.05) in oxidative stress and inflammatory response markers, as measured by phase II antioxidant enzymes and q-PCR gene expression analysis. However, the overall magnitude of responses was low, and oxidative damage was not provoked. Further, a significant elimination of Au from the digestive tract within a 7-day purification period was observed, with excretion being an important pathway. In conclusion, short-term and sub-chronic exposure to an environmental relevant concentration of citrate-stabilized AuNPs cannot be considered toxic to our model organism, while some further consideration should be given to chronic exposure effects.This work was funded by the regional government of Andalusia (Junta de Andalucía) project PE2011-RNM-7812 and the Spanish government Plan Nacional I+D+I project CTM2012-3872-C03-03, as well as supported by the Erasmus Mundus Ph.D. fellowship in Marine and Coastal Management to M. V. (as coordinated by the University of Cadiz, Spain).Peer reviewe