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International audience; Antidepressants in coastal waters may affect ontogeny of predatory behaviour in cuttlefish, which may, as a result, affect growth of newly-hatched cuttlefish. We investigated the effects of two of the most prescribed antidepressants, fluoxetine (FLX) and venlafaxine (VEN) in environmentally realistic concentrations on the predatory behaviour of hatchlings of Sepia officinalis. Newly-hatched cuttlefish were exposed from 1 hour (i.e., day 1) to 5 days after hatching to either FLX alone (5 ng·L-1) combined with VEN (2.5 ng·L-1 or 5 ng·L-1 each) to simulate an environmentally realistic exposure scenario. Their predatory behaviour was analysed through several parameters: prey detection, feeding motivation and success in catching the prey. All parameters improved in control animals over the first five days. The combination of FLX and VEN at 5 ng·L-1 each altered the predatory behaviour of the hatchlings by increasing the latency before attacking the prey, i.e., reducing feeding motivation, as well as by reducing the number of successful attacks. The changes in predatory behaviour tended to reduce food intake and affected growth significantly at 28 days post-hatching. Exposures to either FLX at 5 ng·L-1 or FLX and VEN in mixture at 2.5 ng·L-1 each tended to produce similar effects, even though they were not statistically significant. It is likely that the antidepressants affect maturation of the predatory behaviour and/or learning processes associated with the development of this behaviour. The slightest delay in maturation processes may have detrimental consequences for growth and population fitness.

International audience; Pharmaceuticals such as antidepressants are constantly released into the aquatic environment. Consequently, fluoxetine (FLX) and venlafaxine (VEN), the active molecules of Prozac© and Effexor©, are detected up to several µg.L−1 in freshwater and marine coastal waters. Both compounds act on the serotoninergic system, which may result in behavioural impairment, especially in juvenile animals presumed to be more susceptible to low concentrations than adults. The objective of this study was to determine whether environmental concentrations of FLX alone or combined with VEN modulate innate burying behaviour in two juvenile marine invertebrates, i.e. Sepia officinalis and Carcinus maenas. Juvenile cuttlefish were exposed from hatching to 30 days post-hatching to either FLX alone (i.e. 5 ng.L−1) or in mixture with VEN (i.e. either 2.5 ng.L−1 or 5 ng.L−1 of each antidepressant). Juvenile crabs (

Besides their obvious role in sex determination and reproduction, oestrogens display a prominent and complex immunomodulatory role across all vertebrates. To date, our knowledge on the oestrogenic immunomodulation in non-mammalian species is, however, scarce. In both teleosts and mammals, the direct immunomodulatory function of oestrogen is underscored by the presence of multiple oestrogen receptor subtypes in the various immune cells. For a better understanding of the regulatory processes, we investigated the oestrogen receptor expression in two major lymphoid organs of European sea bass: the head-kidney and the spleen. All oestrogen receptor subtypes, including nuclear and membrane oestrogen receptors, were present in both immune organs as well as in the isolated leucocytes. The same findings have been previously made for the thymus. To determine the oestrogen responsiveness of the different immune cell populations and to evaluate the importance of non-genomic and genomic pathways, we assessed the kinetics and the concentration dependent effects of 17β-oestradiol on isolated leucocytes from the head-kidney, the spleen and the thymus in vitro. Given the importance of reactive oxygen species as signalling and defence components in mammalian immune cells, the oxidative burst capacity, the redox status and the viability of both lymphoid and myeloid cells were measured by flow cytometry. The treatment with 17β-oestradiol specifically modulated these parameters depending on (1) the time kinetic, (2) the concentration of 17β-oestradiol, (3) the immune cell population (lymphoid and myeloid cells) as well as (4) the lymphoid organs from which they originated. The observed in vitro oestrogenic effects as well the presence of various oestrogen receptor subtypes in the immune cells of sea bass suggest a complex and direct oestrogenic action via multiple interconnected oestrogen-signalling pathways. Additionally, our study suggests that the oestrogenic regulation of the sea bass immune function involves a direct and tissue specific modulation of the immune cell redox biology comprising redox signalling, NADPH-oxidase activity and H2O2-permeability, thus changing oxidative burst capacity and immature T cell fate because oestrogen impacted thymocyte viability. Importantly, immune cells from both primary and secondary lymphoid organs have shown specific in vitro oestrogen-responsiveness. As established in mammals, oestrogen is likely to be specifically and directly involved in immature T cell differentiation and mature immunocompetent cell function in sea bass too.

Serotonin (5-HT) takes a key position in regulating vital functions, such as cardio-ventilatory activity, locomotion and behaviour. Selective serotonin reuptake inhibitors (SSRIs) modulate the serotonergic system and thus affect these functions. Rhythmic behaviours, such as cardio-ventilatory activity, are controlled by central pattern generators, which in turn are regulated by 5-HT. In crustaceans, 5-HT also regulates the synthesis and secretion of crustacean hyperglycaemic hormone, a pleiotropic hormone involved in the mobilisation and release of glucose into the haemolymph, thus stimulating the animal’s activity. As a matter of consequence, SSRIs may affect cardio-ventilatory activity. In order to examine how the SSRIs affect fundamental physiological parameters based on rhythmic behaviours in decapods, cardio-respiratory activity in the shore crab Carcinus maenas was assessed after pericardial injection of a single dose of either 0.5 μM, 0.75 μM or 1 μM fluoxetine, respectively. Simultaneous recordings of heart and scaphognathite movements in both brachial chambers were conducted by measuring impedance changes in the respective body compartments. Injection of 5-HT had an immediate effect on cardio-ventilatory activities and strongly upregulated both cardiac and ventilatory activities. Fluoxetine showed similar effects, entailing moderate tachycardia and increased ventilation rates. Compared to 5-HT, these effects were delayed in time and much less pronounced. Metabolism of fluoxetine into the active compound nor-fluoxetine might account for the delayed action, whereas compensatory regulation of cardio-ventilatory frequencies and amplitudes are likely to explain the attenuation of the responses compared to the strong and immediate increase by 5-HT. Overall, the results suggest increased 5-HT levels in invertebrates following fluoxetine exposure, which are able to disturb physiological functions regulated by 5-HT, such as cardiac and respiratory activity.

The disposition and metabolism of fluoxetine in the European shore crab and the Dungeness crab were assessed. Crabs received intracardiac doses of either 0.13 μg/kg or 0.5 mg/kg fluoxetine, respectively. In addition, fluoxetine was administered to Metacarcinus cancer by oral gavage at 7.8 mg/kg. The distribution of fluoxetine was quantified in haemolymph and digestive gland for both crabs, as well as brain, muscle, and testis of Carcinus maenas, over 12 days. The metabolite norfluoxetine, was also measured in C. maenas. Fluoxetine was mainly found in lipid rich tissues. Distribution coefficients increased for digestive gland until three days after fluoxetine administration and then decreased until the end of the observations. The highest distribution coefficients were obtained for brain. Norfluoxetine displayed continuously high levels in digestive gland and brain. The strong decrease in fluoxetine and the concomitant increase in norfluoxetine demonstrates that decapod crustaceans metabolise fluoxetine into the more biologically active norfluoxetine. Fluoxetine levels in the haemolymph of M. cancer declined within 20 h, but showed a second peak 25 h later, suggesting remobilisation from tissues sequestering the compound. The steady state volume distribution and the total body clearance of fluoxetine were high, consistent with high diffusion of fluoxetine into the peripheral tissues and biotransformation as an important elimination pathway. Oral administration of fluoxetine prolonged its half-life in M. cancer, but bioavailability was low. These results confirm the high distribution into nervous tissue, extensive biotransformation into the highly active norfluoxetine and a half-life similar to that observed in vertebrates.

The highly conserved part of the nucleotide-binding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucose-related hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.

The internal temperature of land snails depends on environmental factors, such as exposure to electromagnetic radiation and airflow as well as biotic factors including shell size, shell colouration and thickness or the resting position of the snail. In controlled field experiments, we quantified heating by thermal absorption of light and airflow-induced heat loss in the white garden snail, Theba pisana , from Normandy, France. Heating experiments revealed a significant positive relation of the internal body temperature with illumination period, shell temperature and air temperature at different times of day. The size of the snails was negatively related with both of the given illumination times: smaller animals heated up stronger than larger ones. The temperature at the surface of the shell significantly depended on the illumination period and the time of day. An AIC-based quality assessment of multiple linear modelling showed that, for explaining both shell surface and internal temperature of the soft body, several factors, i.e. , exposure time, daytime, shell size and colouration contributed to the best models, respectively. Similarly, heat loss of the soft body after and during exposure of the snails to sunlight by a constant airflow depended on the initial body temperature, shell size, colouration and ambient air temperature. Our study revealed also the importance of both shell size and colouration for the loss of body temperature under natural conditions: small and banded animals that had heated up to temperatures above 30 °C cooled down faster than large and un-banded ones.

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As a common sessile filter feeder with high bioconcentration rates, Dreissena polymorpha is useful as a bioindicator species. Pollution and environmental factors influence the reproductive effort of these bivalves. Therefore, the evaluation of the gametogenic cycle is important for understanding environmentally-related physiological alterations in this dioecious species. Animals from an unpolluted reference site close to the highly polluted Seine River, France, were monitored monthly over 1 year using qualitative and quantitative measures of gametogenesis. Developmental stages of the gonad were determined by visual inspection of histological slides. Unbiased quantitative stereological analysis was conducted to estimate oocyte development and the gonad index was determined. To investigate a possible role of female sex steroids in bivalve gametogenesis as a possible target for xenoestrogens, free oestradiol levels were assessed and analysed with respect to oocyte development. The basic pattern of the reproductive cycle consisted of early gametogenesis during autumn and winter, followed by rapid vitellogenesis in early spring, and spawning towards the summer months. However, the population was characterized by high inter-individual variability of the gonad stages. Furthermore, the simultaneous occurrence of different developmental stages within the same animal made classification difficult. Stereological determination of oocyte volumes as well as gonadosomatic index provided a more precise evaluation of reproductive effort. Oestradiol levels did vary significantly between months, but did not show any clear relationship with a particular stage of gametogenesis or oocyte volume.

Amongst animals, several hydrogen peroxide-generating oxidases are apparently restricted to molluscs. One of these, d -mannitol oxidase, is concentrated in the alimentary system, where it is associated with its own subcellular membrane system of unique tubular morphology, most likely representing a structural modification of the ER. These structures can be purified by subcellular fractionation and have been termed ‘mannosomes’. Little is known about the functions of mannitol oxidase or of mannosomes, but the previously reported molluscicide-induced increase in mannosomes implies their involvement in a general stress reaction. In this study, we examined the effects of heavy metal stress in the terrestrial gastropod Arion lusitanicus . The activity of mannitol oxidase and mannosome abundance were monitored, together with metal effects on heat-shock protein level, and these parameters were compared to heavy metal accumulation in the digestive gland. We found that mannitol oxidase is inhibited by heavy metals more than other oxidases. On the other hand, hsp70 levels and mannosomal protein were increased with enhanced heavy metal stress, the latter indicating a probable increase in the number of mannosome organelles. Thus, stress protein (hsp70) and mannosomal protein were positively correlated with heavy metal accumulation, whereas the enzyme activity showed a negative correlation with increasing heavy metal content of the slugs.

Within the growing body of proteomics studies, issues addressing problems of ecotoxicology are on the rise. Generally speaking, ecotoxicology uses quantitative expression changes of distinct proteins known to be involved in toxicological responses as biomarkers. Unlike these directed approaches, proteomics examines how multiple expression changes are associated with a contamination that is suspected to be detrimental. Consequently, proteins involved in toxicological responses that have not been described previously may be revealed. Following identification of key proteins indicating exposure or effect, proteomics can potentially be employed in environmental risk assessment. To this end, bioinformatics may unveil protein patterns specific to an environmental stress that would constitute a classifier able to distinguish an exposure from a control state. The combined use of sets of marker proteins associated with a given pollution impact may prove to be more reliable, as they are based not only on a few unique markers which are measured independently, but reflect the complexity of a toxicological response. Such a proteomic pattern might also integrate some of the already established biomarkers of environmental toxicity. Proteomics applications in ecotoxicology may also comprise functional examination of known classes of proteins, such as glutathione transferases or metallothioneins, to elucidate their toxicological responses.

Background Proteomics may help to detect subtle pollution-related changes, such as responses to mixture pollution at low concentrations, where clear signs of toxicity are absent. The challenges associated with the analysis of large-scale multivariate proteomic datasets have been widely discussed in medical research and biomarker discovery. This concept has been introduced to ecotoxicology only recently, so data processing and classification analysis need to be refined before they can be readily applied in biomarker discovery and monitoring studies. Results Data sets obtained from a case study of oil pollution in the Blue mussel were investigated for differential protein expression by retentate chromatography-mass spectrometry and decision tree classification. Different tissues and different settings were used to evaluate classifiers towards their discriminatory power. It was found that, due the intrinsic variability of the data sets, reliable classification of unknown samples could only be achieved on a broad statistical basis (n > 60) with the observed expression changes comprising high statistical significance and sufficient amplitude. The application of stringent criteria to guard against overfitting of the models eventually allowed satisfactory classification for only one of the investigated data sets and settings. Conclusion Machine learning techniques provide a promising approach to process and extract informative expression signatures from high-dimensional mass-spectrometry data. Even though characterisation of the proteins forming the expression signatures would be ideal, knowledge of the specific proteins is not mandatory for effective class discrimination. This may constitute a new biomarker approach in ecotoxicology, where working with organisms, which do not have sequenced genomes render protein identification by database searching problematic. However, data processing has to be critically evaluated and statistical constraints have to be considered before supervised classification algorithms are employed.

Proteomics provide potential in the discovery of new sensitive biomarkers for environmental pollution. To evaluate this potential, we have utilized ProteinChip® technology to analyze the proteomic profile of blue mussels (Mytilus edulis) from polluted marine habitats surrounding the island of Karmoy, Norway. Two different types of contamination, heavy metals and polyaromatic hydrocarbons (PAHs), were compared to a clean reference site. Differentially expressed proteins/peptides were found, which showed a specific induction or a general suppression associated with the field site of origin. By combining sets of protein markers in a tree-building algorithm, we were able to correctly classify samples from these sites with an accuracy of 90%.

International audience; The impact of lead on food consumption, energy metabolism and the stress protein (hsp 70) level was investigated in the woodlouse Porcellio scaber (Isopoda), a common representative of the saprophagous soil macrofauna. To examine possible acclimation or tolerance to lead in woodlice from a contaminated habitat, animals of two populations, one deriving from a lead-contaminated artillery range and one from an uncontaminated control stand, were exposed to a series of lead concentrations under otherwise constant laboratory conditions for a maximum of 80 days. The applied lead concentrations (at a maximum 7945 mg/kg food dry wt) did not have any significant quantitative effect on the food consumption of the isopods, although the population pre-exposed in the artillery range showed a tendency toward a higher food uptake than the control population. After 80 days of exposure, both populations showed an equal trend toward increasing their respiration as lead concentrations, that they had been fed on, were increased. Accordingly, the glycogen content of the body, in both populations, was elevated with increasing lead concentrations in the food. This effect was more pronounced in the pre-exposed isopod population than in the one from the control stand. The non-pre-exposed isopods showed a general tendency toward a lower protein content of their bodies than the pre-exposed ones, although no effect of the lead on this parameter could be statistically proven. The ability of the artillary range isopods to synthesise stress proteins (hsp 70) in response to lead contamination decreased at much lower lead concentrations in their food than in the non-pre-exposed control population, even though the artillery range isopods seemed to be equally or even slightly better equipped with energy storage products. Even though the better nutrient status of these animals might refer to some lead tolerance of the pre-exposed population, the stress protein data suggest that a metal-resistent Porcellio population did not evolve in this lead-contaminated site.

International audience; It has been noted that both summer drought and sub-zero winter temperatures induce the synthesis of sugars and polyols in invertebrate tissues. This has led several authors to suggest that many of the adaptations, previously viewed as a response to cold, might be part of a more universal desiccation tolerance mechanism. Here we show that acclimation of the soil dwelling collembolan Folsomia candida to a sublethal desiccation stress confers tolerance to cold shock and a significant increase in the molar percent of membrane fatty acids with a mid-chain double bond. These changes in membrane fatty acids are interpreted as conferring a significant reduction in the transition temperature of cell membranes, as would be expected in acclimation to cold, and these changes are therefore interpreted as contributing to the cross-tolerance. Drought acclimation was also shown to trigger the synthesis of the 70kDa family of heat-shock proteins (Hsp70). This group of heat shock proteins is implicated in the reestablishment of the normal three-dimensional structure of partially unfolded proteins and therefore are also likely to contribute to the observed cross-tolerance. This study provides evidence that the stresses exerted by desiccation and cold at the cellular level have sufficient similarities to induce overlapping adaptations.