ToF-SIMS imaging shows specific lipophilic vitamin alterations in chronic reprotoxicity caused by the emerging contaminant Pravastatin in Gammarus fossarum.

Blood lipid-lowering agents, such as Pravastatin, are among the most frequently used pharmaceuticals released into the aquatic environment. Although their effects on humans are very well understood, their consequences on freshwater organisms are not well known, especially in chronic exposure conditions. Gammarus fossarum is commonly used as sentinel species in ecotoxicology because of its sensitivity to a wide range of environmental contaminants and the availability of standardized bioassays. Moreover, there is an increased interest in linking molecular changes in sentinel species, such as gammarids, to observed toxic effects. Here, we performed a reproductive toxicity assay on females exposed to different concentrations of pravastatin (30; 300; 3,000 and 30,000 ng L-1) during two successive reproductive cycles and we applied ToF-SIMS imaging to evaluate the effect of pravastatin on lipid homeostasis in gammarids. Reproductive bioassay showed that pravastatin could affect oocyte development in Gammarus fossarum inducing embryotoxicity in the second reproductive cycle. Mass spectrometry imaging highlighted the disruption in vitamin E production in the oocytes of exposed female gammarids at the second reproductive cycle, while limited alterations were observed in other lipid classes, regarding both production and tissue distribution. The results demonstrated the interest of applying spatially resolved lipidomics by mass spectrometry imaging to assess the molecular effects induced by long-term exposure to environmental pharmaceutical residues in sentinel species.

Shotgun lipidomics and mass spectrometry imaging unveil diversity and dynamics in Gammarus fossarum lipid composition.

Sentinel species are playing an indispensable role in monitoring environmental pollution in aquatic ecosystems. Many pollutants found in water prove to be endocrine disrupting chemicals that could cause disruptions in lipid homeostasis in aquatic species. A comprehensive profiling of the lipidome of these species is thus an essential step toward understanding the mechanism of toxicity induced by pollutants. Both the composition and spatial distribution of lipids in freshwater crustacean Gammarus fossarum were extensively examined herein. The baseline lipidome of gammarids of different sex and reproductive stages was established by high throughput shotgun lipidomics. Spatial lipid mapping by high resolution mass spectrometry imaging led to the discovery of sulfate-based lipids in hepatopancreas and their accumulation in mature oocytes. A diverse and dynamic lipid composition in G. fossarum was uncovered, which deepens our understanding of the biochemical changes during development and which could serve as a reference for future ecotoxicological studies.

A "Population Dynamics" Perspective on the Delayed Life-History Effects of Environmental Contaminations: An Illustration with a Preliminary Study of Cadmium Transgenerational Effects over Three Generations in the Crustacean Gammarus.

We explore the delayed consequences of parental exposure to environmentally relevant cadmium concentrations on the life-history traits throughout generations of the freshwater crustacean Gammarus fossarum. We report the preliminary results obtained during a challenging one-year laboratory experiment in this environmental species and propose the use of population modeling to interpret the changes in offspring life-history traits regarding their potential demographic impacts. The main outcome of this first long-term transgenerational assay is that the exposure of spawners during a single gametogenesis cycle (3 weeks) could result in severe cascading effects on the life-history traits along three unexposed offspring generations (one year). Indeed, we observed a decrease in F1 reproductive success, an early onset of F2 offspring puberty with reduced investment in egg yolk reserves, and finally a decrease in the growth rate of F3 juveniles. However, the analysis of these major transgenerational effects by means of a Lefkovitch matrix population model revealed only weak demographic impacts. Population compensatory processes mitigating the demographic consequences of parental exposure seem to drive the modification of life-history traits in offspring generations. This exploratory study sheds light on the role of population mechanisms involved in the demographic regulation of the delayed effects of environmental toxicity in wild populations.

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.

Fenoxycarb exposure disrupted the reproductive success of the amphipod Gammarus fossarum with limited effects on the lipid profile.

Insect growth regulator insecticides mimic the action of hormones on the growth and development of insect pests. However, they can affect the development of non-target arthropods. In the present study, we tested the effects of the growth regulator insecticide fenoxycarb on several endpoints in the freshwater crustacean Gammarus fossarum (Amphipoda). Females carrying embryos in their open brood pouch were exposed to 50 µg L-1 fenoxycarb throughout the entire oogenesis (i.e. 21 days). After exposure, newborn individuals from exposed embryos were removed from the maternal open brood pouch for lipidomic analysis, while males were added to assess the reproductive success. After fertilization, the lipid profile, energy reserve content (lipids, proteins and glycogen), and activity of phenoloxidase - an enzyme involved in the immune response - were measured in females. No significant effect of fenoxycarb exposure was observed on the lipid profile of both newborn individuals and females, while reproductive success was severely impaired in exposed females. Particularly, precopulatory behavior was significantly reduced and fertilized eggs were unviable. This study highlighted the deleterious effects of the insect growth regulator fenoxycarb on gammarid reproduction, which could have severe repercussions on population dynamics.

Phenotypic defects in newborn Gammarus fossarum (Amphipoda) following embryonic exposure to fenoxycarb.

During morphogenesis numerous morphogenetic factors ensure the production of a target phenotype. By disrupting these processes, a toxic exposure during this period could cause an increase of phenotypic defects. In the present study, embryos of the freshwater amphipod Gammarus fossarum were exposed throughout the embryogenesis to increasing concentrations of fenoxycarb (0, 0.5µgL-1, 5µgL-1 and 50µgL-1), a growth regulator insecticide analog of the insect juvenile hormone. In addition, to identify morphogenesis' sensitive period, embryos were exposed during either early or late embryonic development to 5µgL-1 of fenoxycarb. In newborn individuals from exposed embryos, three phenotypes were investigated: i) eye pigmentation, ii) length of the antenna and gnathopod of both left and right sides and iii) midgut tissue state. Developmental homeostasis was assessed by measuring fluctuating asymmetry and inter-individual variance of both the antenna and gnathopod. Exposure to 5µgL-1 and 50µgL-1 fenoxycarb throughout the embryonic development induced a delayed hatching and altered appendages size. Moreover, exposure to 5µgL-1 throughout the embryogenesis and during the gastrulation phase impaired eye pigmentation, while exposure to 50µgL-1 resulted in increased tissue damages of the midgut. No significant increase of fluctuating asymmetry was observed in exposed individuals, neither for the antenna nor for the gnathopod. These results demonstrate that fenoxycarb can alter embryonic development of G. fossarum without disrupting developmental homeostasis.

Gammarus fossarum as a sensitive tool to reveal residual toxicity of treated wastewater effluents.

Wastewater treatment plants (WWTPs) are one of the main sources of freshwater pollution eventually resulting in adverse effects in aquatic organisms. Treated effluents can contain many micropollutants at concentrations often below the limit of chemical quantification. On a regulatory basis, WWTP effluents have to be non-toxic to the aquatic environment, wherefore not only chemical abatement but also ecotoxicological evaluation through relevant bioassays is required. Standardized bioassays currently used are often not sensitive enough to reveal a residual toxicity in treated effluents. Therefore, attention must be paid to the development of better-adapted approaches implementing more sensitive organisms and relevant endpoints. In this study, the toxicity of two differently treated effluents (activated sludge treated effluents with and without ozonation) towards the ecologically relevant species Gammarus fossarum was evaluated. Organism fitness traits such as reproduction and sperm DNA integrity were followed in exposed organisms. In complement, enzymatic biomarkers were measured indicating the presence of neurotoxic compounds (acetylcholinesterase activity), the presence of pathogens likely to increase the toxic effects of chemical compounds (phenol-oxidase activity), and the presence of toxic compounds inducing detoxification mechanisms (glutathione-S-transferase activity). Enzymatic activities were not modified, but significant sub-lethal effects were observed in exposed organisms. In both effluents, females showed a retarded molt cycle, a reduced fecundity and fertility, and >90% of developed embryos exhibited developmental malformations. In addition, a slight but significant genotoxic effect was measured in gammarid sperm. In a whole, no difference in toxicity was found between both effluents. Coupling reproduction impairment and genotoxicity assessment in Gammarus fossarum seems to be a valuable and sensitive tool to reveal residual toxicity in effluents containing a mixture of micropollutants at very low concentrations. Finally, a direct relationship between the observed toxic responses and the quantified micropollutant concentrations could not be evidenced.

Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.

The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly - regardless the time-intervals - at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight "day" effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the "day" effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails' immunocompetence.

Impact of cadmium on the ecdysteroids production in Gammarus fossarum.

Gammarus fossarum is an important test organism which is currently used as a bio-indicator as well as in ecotoxicological tests. Nevertheless, data on ecdysteroids in endocrine toxicity test are not yet available for these species, despite its crucial role in molting and reproduction. In the present paper, ecdysteroids concentrations were studied during the molt cycle (in females) and embryonic development in G. fossarum (Crustacea, Amphipoda) in order to propose an ecdysteroids toxicity test. Ecdysteroids levels in G. fossarum showed a single peak during premolt at stage Dl-D2. In embryos, ecdysteroids levels progressively increased over stages 3 and 4, with peak levels at stage 4. A Cadmium toxicity test was proposed to examine if the molting and embryogenesis disturbances previously observed after cadmium exposure (Geffard et al. 2010) could be attributed to changes in ecdysteroids titers. Exposure to the different cadmium concentrations (3; 9; 300; 900 µg/l) increased ecdysteroids secretion by Y-organs in vitro, but it had no significant effect on exposed embryos (in vivo). Based on previous findings, we are led to conclude that the molting impairments in cadmium-exposed females of G. fossarum is connected to the changes in ecdysteroids concentrations.

Proteomic investigation of male Gammarus fossarum, a freshwater crustacean, in response to endocrine disruptors.

While the decrease in human sperm count in response to pollutants is a worldwide concern, little attention is being devoted to its causes and occurrence in the biodiversity of the animal kingdom. Arthropoda is the most species-rich phyla, inhabiting all aquatic and terrestrial ecosystems. During evolution, key molecular players of the arthropod endocrine system have diverged from the vertebrate counterparts. Consequently, arthropods may have different sensitivities toward endocrine disrupting chemicals (EDCs). Here alteration of sperm quality in a crustacean, Gammarus fossarum, a popular organism in freshwater risk assessment, was investigated after laboratory exposure to various concentrations of three different xenobiotics: cadmium, methoxyfenozide, and pyriproxyfen. The integrity of the reproductive process was assessed by means of sperm-quality markers. For each substance, semiquantitative/relative proteomics based on spectral counting procedure was carried out on male gonads to observe the biological impact. The changes in a total of 871 proteins were monitored in response to toxic pressure. A drastic effect was observed on spermatozoon production, with a dose-response relationship. While exposure to EDCs leads to strong modulations of male-specific proteins in testis, no induction of female-specific proteins was noted. Also, a significant portion of orphans proved to be sensitive to toxic stress.

Exposure of juvenile Danio rerio to aged TiO2 nanomaterial from sunscreen.

The toxicity of dietary exposure to artificially aged TiO2 nanomaterial (T-Lite) used in sunscreen cream was studied on Danio rerio. Embryolarval assays were conducted to assess the effects of TiO2 residues of nanomaterial (RNM) on fish early life stages. Juvenile fishes were exposed by the trophic route in two experiments. During the first experiment, juvenile fishes were exposed to TiO2 RNM for 14 days by adding RNM to commercial fish food. The second one consisted in producing a trophic food chain. Pseudokirchneriella subcapitata algae, previously contaminated with TiO2 RNM in growth medium, was used to feed Daphnia magna neonates over a 48-h period. Daphnia were used next to feed juvenile fishes for 7 days. Accumulation of Ti, life traits (survival and growth) and biochemical parameters such as energy reserves, digestive (trypsin, esterase, cellulose and amylase) and antioxidant (superoxide dismutase and catalase) enzyme activity were measured at the end of exposures. As expected in the receiving aquatic system, TiO2 RNM at low concentrations caused a low impact on juvenile zebrafish. A slight impact on the early life stage of zebrafish with premature hatching was observed, and this effect appeared mainly indirect, due to possible embryo hypoxia. When juvenile fish are exposed to contaminated food, digestive enzyme activity indicated a negative effect of TiO2 RNM. Digestive physiology was altered after 14 days of exposure and seemed to be an indirect target of TiO2 RNM when provided by food.

Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment.

Among freshwater invertebrates, Gammarus fossarum is an important test organism and is currently used in ecotoxicology for acute and chronic assays; nevertheless, reproductive toxicity test methods are not yet available for these species. In the present study, the reproductive cycle in Gammarus fossarum was characterized in order to propose a reproductive toxicity test encompassing molting, follicle growth, and embryonic development that will provide a better understanding of the mode of action of chemicals disrupting these hormone-regulated processes. A detailed description of the reproductive cycle in Gammarus fossarum was obtained. As in some amphipods, molt and reproductive cycles of G. fossarum females occur concurrently, lasting 30 d at 12°C. Each molt stage is characterized by a specific marsupial embryonic development stage and the size of developing follicles visible on the ovarian membrane. Based on these results, a 21-d reproductive toxicity test is proposed for this species. This new bioassay was applied to identify the specific impact of different stressors: cadmium, methomyl, nonylphenol, and a starvation diet. Good reproducibility was obtained for different endpoints under control conditions and throughout the experiments. Preliminary robust reference values or benchmarks were proposed for these endpoints. Cadmium was found to specially inhibit secondary vitellogenesis. Nonylphenol had a specific concentration-dependent effect on embryonic development, with an increase in the percent abnormality from a concentration of 0.05 µg/L. A restricted food diet led to a significant delay in the molt cycle, which in turn induced inhibition of secondary vitellogenesis.

Development of biomarkers of stress related to endocrine disruption in gastropods: alkali-labile phosphates, protein-bound lipids and vitellogenin-like proteins.

The occurrence in aquatic environments of compounds capable of acting as endocrine disruptors (ED) is of concern for wildlife. Elevated levels of the egg-yolk precursor protein vitellogenin (Vg) are widely accepted as a biomarker for xenoestrogen exposure in aquatic organisms. The purpose of this study was to develop a method for tracking changes in levels of Vg-like proteins in gastropods. Vg-like and egg-yolk proteins were analyzed in three freshwater gastropods having different modes of reproduction: Potamopyrgus antipodarum (asexual reproduction), Valvata piscinalis (hermaphroditism) and Lithoglyphus naticoides (sexual reproduction). Vitellogenin-like protein levels were examined by the alkali-labile phosphate (ALP) technique, a novel protein-bound lipid (PBL) assay, and by gradient gel electrophoresis with silver staining. The first phase of the study was dedicated to the development and optimization of an analytical method for detecting Vg-like proteins in these three gastropod species. In the second phase, the snails P. antipodarum and V. piscinalis were exposed to bisphenol A (BPA), octylphenol (OP) and tributyltin (TBT) for 14 and 28 days. Vg-like proteins were resolved in one major band at 250-300 kDa for L. naticoides and V. piscinalis and in two bands at 100 and 30 kDa for P. antipodarum. After 14 days of exposure, all techniques showed an increase in Vg-like protein levels at 100 microg/L BPA and at 1 microg/L OP in P. antipodarum. A decrease in these proteins was observed with high concentrations of OP (100 microg/L) and TBT (>or=5 ng/L). In V. piscinalis, a decrease in Vg-like proteins was shown after 14 days of exposure for OP >or=10 microg/L and TBT >or=5 ng/L; however, at 28 days, gel electrophoresis revealed an increase in these proteins. Histological observations showed significant necrosis in ovotestes of V. piscinalis with the three endocrine-disrupting compounds, while tissue modifications were not detected for P. antipodarum. A method for measuring Vg-like and egg-yolk proteins is proposed to track changes in both estrogenic and androgenic endocrine disruptors in gastropods. The potential use of these species as bioindicators of endocrine disruption in freshwater environments in terms of their reproduction mode is discussed.

Cloning, expression and regulation of chicken ovalbumin upstream promoter transcription factors (COUP-TFII and EAR-2) in the rat anterior pituitary gland.

Chicken ovalbumin upstream promoter transcription factors (COUP-TF)-II (NR2F2) and EAR-2 (NR2F6) are structurally related orphan members of the nuclear receptors superfamily. There are growing evidences that these factors play important roles during processes of differentiation and proliferation of several tissues. To better understand their role in the differentiated adult rat pituitary gland, we cloned COUP-TFII and EAR-2 cDNAs from an anterior pituitary cDNA library. Subsequently, we raised and characterized specific antibodies to the N-terminal domain of both nuclear receptors. We next examined their cellular and subcellular distribution in the pituitary gland and determined their regulation during pregnancy. COUP-TFII and EAR-2 pituitary genes display, respectively, 90 and 100% homologies with their human and mouse homologues. Cellular expression of both nuclear receptors was mainly detected in the lactotropes of male and female rats, with a prominent distribution in the nuclear compartment for EAR-2, and interestingly both proteins were significantly upregulated in pituitaries of pregnant vs. cycling female rats. Thus, our results have characterized cloning of rat pituitary COUP-TFII and EAR-2 genes, demonstrated that they are both specifically expressed in lactotropes, and strongly suggested that they may play an important role in modulating prolactin (PRL) gene expression during pregnancy.