Comparison in the response of three European Gammarid species exposed to the growth regulator insecticide fenoxycarb.

Growth regulator insecticides with juvenoid activity can affect the development and reproduction of non-target organisms such as crustaceans. In this perspective, our previous studies revealed deleterious effects of the juvenoid fenoxycarb at 5 µg L-1 on the embryogenesis and at 50 µg L-1 on the reproductive behavior of the amphipod Gammarus fossarum. In the present study, to determine whether data generated with one amphipod species can be extended to other gammarid species, we tested the effects of a 5 µg L-1 fenoxycarb exposure on three European amphipod species: G. fossarum, Gammarus roeseli, and Echinogammarus longisetosus. We exposed individually 60 freshly fertilized females to fenoxycarb throughout the entire oogenesis/embryogenesis cycle (i.e., 19 days). In newborn individuals from exposed embryos, we measured both pigmentation and lipid reserve impairments while in exposed females, we observed reproductive behavior. At 5 µg L-1 fenoxycarb, reproductive behavior was only altered in G. fossarum. This study demonstrates the variability of the toxic response among the three gammaridae species, underlining the need for acquiring data with a broad phylogenetic representation to better predict toxic effects on freshwater ecosystems.

Vitellogenin-like proteins among invertebrate species diversity: potential of proteomic mass spectrometry for biomarker development.

Cost-effective methodologies along with cross-species applicability constitute key points for biomarker development in ecotoxicology. With the advent of cheaper affordable genomic techniques and high throughput sequencing, omics tools could facilitate the assessment of effects of environmental contaminants for all taxa biodiversity. We assessed the potential of absolute quantification of proteins using mass spectrometry to develop vitellogenin (Vg)-like protein assays for invertebrates. We used available sequences in public databases to rapidly identify Vg-proteotypic peptides in seven species from different main taxa of protostome invertebrates (mollusk bivalves, crustacean amphipods, branchiopods, copepods and isopods, and insect diptera). Functional validation was performed by comparing proteomic signals from reproductive female tissue samples and negative controls (male or juvenile tissues). In a second part, we demonstrate in gammarids, daphnids, drosophilids, and gastropods that the assay validated in Vg-sequenced species can be applied to Vg-unsequenced species thanks to the evolutionary conservation of Vg-proteotypic peptide motifs. Finally, we discuss the relevance of mass spectrometry for biomarker development (specific measurement, rapid development, transferability across species). Our study supplies an illustration of the promising strategy to address the challenge of biodiversity in ecotoxicology, which consists in employing omics tools from comparative and evolutionary perspectives.

Vitellogenin-like gene expression in freshwater amphipod Gammarus fossarum (Koch, 1835): functional characterization in females and potential for use as an endocrine disruption biomarker in males.

The induction of vitellogenin (Vtg) synthesis is widely accepted as a biomarker of estrogenic exposure in male and juvenile fish. Vtg synthesis has emerged as an interesting endpoint to assess endocrine disruptor (ED) effects in crustaceans. However, studies reporting induction of Vtg in male crustaceans are lacking. This study investigated the expression of the Vtg gene in a freshwater amphipod, Gammarus fossarum, using calibrated real-time reverse transcription polymerase chain reaction (real-time RT PCR). First, we described the basal pattern of expression in healthy male and female organisms at different reproductive moult stages, in order to validate the function of this gene. Females expressed from 200 to 700 times more Vtg transcripts than males, depending on the female reproductive stage. Females displayed significant elevation of Vtg mRNA levels at the end of the inter-moult phase and at the beginning of the pre-moult phase. Second, male gammarids were exposed to the estrogenic compound nonylphenol (NP) (0.05, 0.5, 5 and 50 µg L(-1)) and to the anti-androgen cyproterone (1, 10, 100 and 1000 µg L(-1)) for 2, 4, 8 and 16 days. Both chemicals altered the pattern of interindividual variability of Vtg gene expression in males with strong induction in some individuals. Finally, the impact of urban wastewater treatment plants (WWTP) on male Vtg gene expression was assessed in organisms transplanted in the field during in situ bioassay campaigns in three different watersheds. Induction of the Vtg mRNA level was observed in males transplanted downstream from WWTP effluent discharge in two of the three study sites.

A pollution-monitoring pilot study involving contaminant and biomarker measurements in the Seine Estuary, France, using zebra mussels (Dreissena polymorpha).

Zebra mussel (Dreissena polymorpha) is an invasive species that has proliferated in European and North American rivers and lakes during the last century. In this study, D. polymorpha has been used to provide information on contamination levels and biological effects in the Seine Estuary (France). The bivalves accumulated polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) to a high degree with values reaching 800 ng/g dry weight for PCBs (sum of 20 congeners), and 1,000 ng/g dry weight of PAHs (sum of 14 compounds) in the whole body. These values are among the highest reported of PCBs and, to a lesser extent, of PAHs in other contaminated areas in the world. Toxic equivalent quantities of PCBs and PAHs detected in zebra mussels varied from 20 to 40 pg dioxin equivalents/g dry weight for PCBs and up to 120 ng benzo[a]pyrene equivalents/ g dry weight for PAHs, indicating a high potential risk for animals feeding on them. Biological impacts, such as altered condition index, decreased lysosomal stability, and high levels of multixenobiotic resistance (MXR) proteins also were detected in mussels living downstream of Rouen, the main city of the Seine Estuary. Taken together, these results indicate that the Seine Estuary is a heavily polluted area with the potential to cause deleterious health effects in some endogenous living organisms. This study also shows that chemical and biological measurements bring different but complementary results that can help diagnose environmental health.

Rhodamine exclusion activity in primary cultured turbot (Scophthalmus maximus) hepatocytes.

Cellular detoxification by direct processes has been investigated in fish by studying the ability of hepatocytes prepared from juvenile aquarium-reared turbot (Scophthalmus maximus) to actively exclude the fluorescent dye rhodamine B (RB). Cell viability was studied by measurements of non-specific esterase activity using fluorescein diacetate. This revealed that turbot hepatocytes can be cultured for a few days with a viability decreasing to 38% after 24 h. The 24-h cultured cells have been used to study the rhodamine B exclusion activity using confocal laser microscopy. Hepatocytes accumulated the dye in a competitive manner with verapamil, thus suggesting that they express a transport system similar to the P-glycoprotein-mediated multixenobiotic resistance process. Incubation of cells with 1 microM RB and 20 microM verapamil led to a 26% increase of cellular fluorescence as compared to the accumulation in absence of competitor. Rhodamine B accumulated in the whole cytoplasm, with more concentrated areas that might correspond to the lysosomal compartment and the cell membrane.

Genetic and immunological characterisation of a multixenobiotic resistance system in the turbot (Scophthalmus maximus).

Pleiotropic resistance driven by transport proteins constitutes a very ubiquitous protection mechanism against natural or synthetic toxic compounds. The multidrug (MDR) or multixenobiotic (MXR) system has been identified in many different species, and may be used as a biomarker for pollution assessment. Here we report the existence of a gene encoding a MXR-related protein in a benthic fish species, the turbot Scophthalmus maximus, and its constitutive expression in several tissues. A 433bp cDNA fragment has been cloned by RT-PCR. The deduced amino-acid sequence shares close to 80% homology with class I or class II mammalian MDR proteins. This cDNA corresponds to a major mRNA of 5.6 kb and encodes a protein having an apparent molecular weight of 83 kDa. Constitutive expression levels assessed by semi-quantitative RT-PCR and Western blot, revealed that the kidney and the brain, and to a lesser extent, the heart, gills and intestine, are the organs which contain the highest amount of both MXR mRNAs or proteins. This tissue specific expression suggests a role for the identified mechanism in protection against endogenous or exogenous toxic compounds.