The Dicentrarchus labrax estrogen screen test: A relevant tool to screen estrogen-like endocrine disrupting chemicals in the aquatic environment.

In response to the need for the diversification of regulatory bioassays to screen estrogen-like endocrine disrupting chemical (EEDC) in the environment, we propose the use of a reporter gene assay involving all nuclear estrogen receptors from Dicentrarchus labrax (i.e., sbEsr1, sbEsr2a, or sbEsr2b). Named DLES test (D. labrax estrogen screen), it aims at complementing existing standardized in vitro tests by implementing more estrogen receptors notably those that do not originate from humans. Positive responses were obtained with all three estrogen receptors, and-consistently with observations from other species-variations in sensitivity to E2 were measured. Sensitivity and EC50 values could be classified as follows: sbEsr2b < sbEsr2a < sbEsr1. The pharmacological characterization with a human estrogen receptor antagonist (fulvestrant) successfully validated the specific involvement of each sbEsr and evidenced the capacity of the DLES test to highlight antagonist interactions. The DLES test was applied to WWTP contaminant extracts. A positive response was detected in the inflow sample in accordance with the YES test, but not in the outflow sample. Notwithstanding, the DLES test (sbEsr2b) exhibited greater sensitivity for the screening of those samples. This study demonstrates the need for more comprehensive testing including representatives of marine species for a better detection of EEDCs. The DLES test appears as a pertinent tool to predict adverse effects and to widen the scope of screening and hazard assessment of EEDCs in the environment.

Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bass.

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors.

Proteomic changes in the extracellular environment of sea bass thymocytes exposed to 17α-ethinylestradiol in vitro.

The thymus is an important immune organ providing the necessary microenvironment for the development of a diverse, self-tolerant T cell repertoire, which is selected to allow for the recognition of foreign antigens while avoiding self-reactivity. Thymus function and activity are known to be regulated by sex steroid hormones, such as oestrogen, leading to sexual dimorphisms in immunocompetence between males and females. The oestrogenic modulation of the thymic function provides a potential target for environmental oestrogens, such as 17α-ethynylestradiol (EE2), to interfere with the cross-talk between the endocrine and the immune system. Oestrogen receptors have been identified on thymocytes and the thymic microenvironment, but it is unclear how oestrogens regulate thymic epithelial and T cell communication including paracrine signalling. Much less is known regarding intrathymic signalling in fish. Secretomics allows for the analysis of complex mixtures of immunomodulatory signalling factors secreted by T cells. Thus, in the present study, isolated thymocytes of the European sea bass, Dicentrarchus labrax, were exposed in vitro to 30 nM EE2 for 4 h and the T cell-secretome (i.e., extracellular proteome) was analysed by quantitative label-free mass-spectrometry. Progenesis revealed a total of 111 proteins differentially displayed between EE2-treated and control thymocytes at an α-level of 5% and a 1.3-fold change cut off (n = 5-6). The EE2-treatment significantly decreased the level of 90 proteins. Gene ontology revealed the proteasome to be the most impacted pathway. In contrast, the abundance of 21 proteins was significantly increased, with cathepsins showing the highest level of induction. However, no particular molecular pathway was significantly altered for these upregulated proteins. To the best of our knowledge, this work represents the first study of the secretome of the fish thymus exposed to the environmental oestrogen EE2, highlighting the impact on putative signalling pathways linked to immune surveillance, which may be of crucial importance for fish health and defence against pathogens.

Prepubertal gonad investment modulates thymus function: evidence in a teleost fish.

Thymus plasticity following gonadectomy or sex hormone replacement has long since exemplified sex hormone effects on the immune system in mammals and, to a lesser extent, in 'lower vertebrates', including amphibians and fish. Nevertheless, the underlying physiological significances as well as the ontogenetic establishment of this crosstalk remain largely unknown. Here, we used a teleost fish, the European sea bass, Dicentrarchus labrax, to investigate: (1) whether the regulation of thymus plasticity relies on resource trade-off with somatic growth and reproductive investment and (2) if the gonad-thymus interaction takes place during gonadal differentiation and development. Because gonadal development and, supposedly, thymus function in sea bass depend on environmental changes associated with the winter season, we evaluated thymus changes (foxn1 expression, and thymocyte and T cell content) in juvenile D. labrax raised for 1 year under either constant or fluctuating photoperiod and temperature. Importantly, in both conditions, intensive gonadal development following sex differentiation coincided with a halt of thymus growth, while somatic growth continued. To the best of our knowledge, this is the first study showing that gonadal development during prepuberty regulates thymus plasticity. This finding may provide an explanation for the initiation of the thymus involution related to ageing in mammals. Comparing fixed and variable environmental conditions, our work also demonstrates that the extent of the effects on the thymus, which are related to reproduction, depend on ecophysiological conditions, rather than being directly related to sexual maturity and sex hormone levels.

Prepubertal gonad investment modulates thymus function: evidence in a teleost fish.

Thymus plasticity following gonadectomy or sex hormone replacement has long since exemplified sex hormone effects on the immune system in mammals and, to a lesser extent, in 'lower vertebrates', including amphibians and fish. Nevertheless, the underlying physiological significances as well as the ontogenetic establishment of this crosstalk remain largely unknown. Here, we used a teleost fish, the European sea bass, Dicentrarchus labrax, to investigate: (1) whether the regulation of thymus plasticity relies on resource trade-off with somatic growth and reproductive investment and (2) if the gonad-thymus interaction takes place during gonadal differentiation and development. Because gonadal development and, supposedly, thymus function in sea bass depend on environmental changes associated with the winter season, we evaluated thymus changes (foxn1 expression, and thymocyte and T cell content) in juvenile D. labrax raised for 1 year under either constant or fluctuating photoperiod and temperature. Importantly, in both conditions, intensive gonadal development following sex differentiation coincided with a halt of thymus growth, while somatic growth continued. To the best of our knowledge, this is the first study showing that gonadal development during prepuberty regulates thymus plasticity. This finding may provide an explanation for the initiation of the thymus involution related to ageing in mammals. Comparing fixed and variable environmental conditions, our work also demonstrates that the extent of the effects on the thymus, which are related to reproduction, depend on ecophysiological conditions, rather than being directly related to sexual maturity and sex hormone levels.

The influence of 17ß-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labrax.

The female sex steroid 17ß-oestradiol (E2) is involved in the regulation of numerous physiological functions, including the immune system development and performance. The role of oestrogens during ontogenesis is, however, not well studied. In rodents and fish, thymus maturation appears to be oestrogen-dependent. Nevertheless, little is known about the function of oestrogen in immune system development. To further the understanding of the role of oestrogens in fish immune system ontogenesis, fingerlings of European sea bass (Dicentrarchus labrax) were exposed for 30 days to 20 ng E2·L-1, at two ages tightly related to thymic maturation, i.e., 60 or 90 days post hatch (dph). The expression of nuclear and membrane oestrogen receptors was measured in the thymus and spleen, and the expression of several T cell-related gene markers was studied in both immune organs, as well as in the liver. Waterborne E2-exposure at 20.2 ± 2.1 (S.E.) ng·L-1 was confirmed by radioimmunoassay, leading to significantly higher E2-contents in the liver of exposed fish. The majority of gene markers presented age-dependent dynamics in at least one of the organs, confirming thymus maturation, but also suggesting a critical ontogenetic window for the implementation of liver resident γδ and αß T cells. The oestrogen receptors, however, remained unchanged over the age and treatment comparisons with the exception of esr2b, which was modulated by E2 in the younger cohort and increased its expression with age in the thymus of the older cohort, as did the membrane oestrogen receptor gpera. These results confirm that oestrogen-signalling is involved in thymus maturation in European sea bass, as it is in mammals. This suggests that esr2b and gpera play key roles during thymus ontogenesis, particularly during medulla maturation. In contrast, the spleen expressed low or non-detectable levels of oestrogen receptors. The E2-exposure decreased the expression of tcrγ in the liver in the cohort exposed from 93 to 122 dph, but not the expression of any other immune-related gene analysed. These results indicate that the proliferation/migration of these innate-like T cell populations is oestrogen-sensitive. In regard to the apparent prominent role of oestrogen-signalling in the late thymus maturation stage, the thymic differentiation of the corresponding subpopulations of T cells might be regulated by oestrogen. To the best of our knowledge, this is the first study investigating the dynamics of both nuclear and membrane oestrogen receptors in specific immune organs in a teleost fish at very early stages of immune system development as well as to examine thymic function in sea bass after an exposure to E2 during ontogenesis.

In vivo effects of serotonin and fluoxetine on cardio-ventilatory functions in the shore crab Carcinus maenas (L. 1758).

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.

Oestrogen differentially modulates lymphoid and myeloid cells of the European sea bass in vitro by specifically regulating their redox biology.

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 17ß-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 17ß-oestradiol specifically modulated these parameters depending on (1) the time kinetic, (2) the concentration of 17ß-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.

Oestrogen, an evolutionary conserved regulator of T cell differentiation and immune tolerance in jawed vertebrates?

In teleosts, as in mammals, the immune system is tightly regulated by sexual steroid hormones, such as oestrogens. We investigated the effects of 17ß-oestradiol on the expression of several genes related to T cell development and resulting T cell subpopulations in sea bass, Dicentrarchus labrax, for a primary lymphoid organ, the thymus, and two secondary lymphoid organs, the head-kidney and the spleen. In parallel, the oxidative burst capacity was assessed in leucocytes of the secondary lymphoid organs. Apoptosis- and proliferation-related genes, indicative of B and T cell clonal selection and lymphoid progenitor activity, were not affected by elevated oestrogen-levels. Sex-related oestrogen-responsiveness in T cell and antigen-presenting cell markers was observed, the expression of which was differentially induced by oestrogen-exposure in the three lymphoid organs. Remarkably, in the spleen, oestrogen increased regulatory T cell-related gene expression was associated with a decrease in oxidative burst capacity. To the best of our knowledge, this study indicates for the first time that physiological levels of oestrogen are likely to promote immune tolerance by modulating thymic function (i.e., T cell development and output) and peripheral T cells in teleosts, similar to previously reported oestrogenic effects in mammals.

Relevance of body size and shell colouration for thermal absorption and heat loss in white garden snails, Theba pisana (Helicidae), from Northern France.

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.

Toxicokinetics, disposition and metabolism of fluoxetine in crabs.

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.

Oestrogen receptor distribution related to functional thymus anatomy of the European sea bass, Dicentrarchus labrax.

In jawed vertebrates, the crosstalk between immune and endocrine system as well as many fundamental mechanisms of T cell development are evolutionary conserved. Oestrogens affect mammalian thymic function and plasticity, but the mechanisms of action and the oestrogen receptors involved remain unclear. To corroborate the oestrogenic regulation of thymic function in teleosts and to identify the implicated oestrogen receptor subtypes, we examined the distribution of nuclear and membrane oestrogen receptors within the thymus of the European Sea bass, Dicentrarchus labrax, in relation to its morpho-functional organisation. Immunohistological analysis specified thymus histology and organisation in teleosts and described, for the first time, Hassall's corpuscle like structures in the medulla of sea bass. All oestrogen receptors were expressed at the transcript and protein level, both in T cells and in stromal cells belonging to specific functional areas. These observations suggest complex regulatory actions of oestrogen on thymic function, notably through the stromal microenvironment, comprising both, genomic and non-genomic pathways that are likely to affect T cell maturation and trafficking processes. Comparison with birds, rodents and humans supports the thymic localization of oestrogen receptors and suggests that oestrogens modulate T cell maturation in all gnathostomes.

Neuroendocrine disruption in the shore crab Carcinus maenas: Effects of serotonin and fluoxetine on chh- and mih-gene expression, glycaemia and ecdysteroid levels.

Serotonin, a highly conserved neurotransmitter, controls many biological functions in vertebrates, but also in invertebrates. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are commonly used in human medication to ease depression by affecting serotonin levels. Their residues and metabolites can be detected in the aquatic environment and its biota. They may also alter serotonin levels in aquatic invertebrates, thereby perturbing physiological functions. To investigate whether such perturbations can indeed be expected, shore crabs (Carcinus maenas) were injected either with serotonin, fluoxetine or a combination of both. Dose-dependent effects of fluoxetine ranging from 250 to 750nM were investigated. Gene expression of crustacean hyperglycemic hormone (chh) as well as moult inhibiting hormone (mih) was assessed by RT-qPCR at 2h and 12h after injection. Glucose and ecdysteroid levels in the haemolymph were monitored in regular intervals until 12h. Serotonin led to a rapid increase of chh and mih expression. On the contrary, fluoxetine only affected chh and mih expression after several hours, but kept expression levels significantly elevated. Correspondingly, serotonin rapidly increased glycaemia, which returned to normal or below normal levels after 12h. Fluoxetine, however, resulted in a persistent low-level increase of glycaemia, notably during the period when negative feedback regulation reduced glycaemia in the serotonin treated animals. Ecdysteroid levels were significantly decreased by serotonin and fluoxetine, with the latter showing less pronounced and less rapid, but longer lasting effects. Impacts of fluoxetine on glycaemia and ecdysteroids were mostly observed at higher doses (500 and 750nM) and affected principally the response dynamics, but not the amplitude of glycaemia and ecdysteroid-levels. These results suggest that psychoactive drugs are able to disrupt neuroendocrine control in decapod crustaceans, as they interfere with the normal regulation of the serotonergic system.

Sensitive periods for 17ß-estradiol exposure during immune system development in sea bass head kidney.

An increasing body of evidence suggests that sex steroids play an important role in the development and regulation of vertebrate immune defense. Therefore, compounds with estrogenic activity may influence the immune system via receptor-mediated pathways. The presence of estrogen receptors in immune cells and organs during the early stages of development may indicate that female steroid hormones are involved in the maturation of the fish immune system. This is of particular importance, as some marine fish are probably exposed to sources of exogenous estrogens while they reside in their estuarine nursery grounds. In this study, the influence of 17ß-estradiol (E2) on estrogen receptor and cytokine gene expression was assessed in juvenile sea bass (Dicentrarchus labrax) together with characterization of the head kidney leukocyte populations and corresponding phagocytic activity during organ regionalization from 98 to 239 dph. E2 exposure, beginning at 90 dph resulted in indirect and delayed modifications of interleukin 1ß and estrogen receptor α gene expression, which may affect B-lymphocyte proliferation in the sea bass head kidney. The E2 treatment of 120 dph fish led to an increase in estrogen receptor ß2 and a decrease in transforming growth factor ß1 gene expression, which coincided with decreased phagocytic activity of head kidney lymphocytes and monocytes/macrophages. Additionally, these changes were observed during developmental periods described as critical phases for B-lymphocyte development in mammals. Consequently, exogenous estrogens have the potential to modify the innate immune response in juvenile sea bass and to exert detrimental effects on head kidney development. Copyright © 2015 John Wiley & Sons, Ltd.

2-DE Mapping of the Blue Mussel Gill Proteome: The Usual Suspects Revisited.

The Blue Mussel (Mytilus edulis, L. 1758) is an ecologically important and commercially relevant bivalve. Because of its ability to bioconcentrate xenobiotics, it is also a widespread sentinel species for environmental pollution, which has been used in ecotoxicological studies for biomarker assessment. Consequently, numerous proteomics studies have been carried out in various research contexts using mussels of the genus Mytilus, which intended to improve our understanding of complex physiological processes related to reproduction, adaptation to physical stressors or shell formation and for biomarker discovery. Differential-display 2-DE proteomics relies on an extensive knowledge of the proteome with as many proteoforms identified as possible. To this end, extensive characterization of proteins was performed in order to increase our knowledge of the Mytilus gill proteome. On average, 700 spots were detected on 2-DE gels by colloidal blue staining, of which 122 different, non-redundant proteins comprising 203 proteoforms could be identified by tandem mass spectrometry. These proteins could be attributed to four major categories: (i) "metabolism", including antioxidant defence and degradation of xenobiotics; (ii) "genetic information processing", comprising transcription and translation as well as folding, sorting, repair and degradation; (iii) "cellular processes", such as cell motility, transport and catabolism; (iv) "environmental information processing", including signal transduction and signalling molecules and interaction. The role of cytoskeleton proteins, energetic metabolism, chaperones/stress proteins, protein trafficking and the proteasome are discussed in the light of the exigencies of the intertidal environment, leading to an enhanced stress response, as well as the structural and physiological particularities of the bivalve gill tissue.

Phenotypic diversity, population structure and stress protein-based capacitoring in populations of Xeropicta derbentina, a heat-tolerant land snail species.

The shell colour of many pulmonate land snail species is highly diverse. Besides a genetic basis, environmentally triggered epigenetic mechanisms including stress proteins as evolutionary capacitors are thought to influence such phenotypic diversity. In this study, we investigated the relationship of stress protein (Hsp70) levels with temperature stress tolerance, population structure and phenotypic diversity within and among different populations of a xerophilic Mediterranean snail species (Xeropicta derbentina). Hsp70 levels varied considerably among populations, and were significantly associated with shell colour diversity: individuals in populations exhibiting low diversity expressed higher Hsp70 levels both constitutively and under heat stress than those of phenotypically diverse populations. In contrast, population structure (cytochrome c oxidase subunit I gene) did not correlate with phenotypic diversity. However, genetic parameters (both within and among population differences) were able to explain variation in Hsp70 induction at elevated but non-pathologic temperatures. Our observation that (1) population structure had a high explanatory potential for Hsp70 induction and that (2) Hsp70 levels, in turn, correlated with phenotypic diversity while (3) population structure and phenotypic diversity failed to correlate provides empirical evidence for Hsp70 to act as a mediator between genotypic variation and phenotype and thus for chaperone-driven evolutionary capacitance in natural populations.

Sample preparation for two-dimensional gel electrophoresis: considering the composition of biological material.

Comparative proteomic analyses in ecotoxicology and related fields require reproducible display of as many proteins as possible. In addition, it should be possible to carry out a quantitative comparison in a reliable manner. Sample preparation represents one of the essential steps toward these aims. In their work, Wu et al. describe how to deal with different recalcitrant tissues of varying species (Proteomics 2013, 13, 3205-3210). Their work underlines the necessity to adapt sample preparation to the specific requirements of the biological material. Beyond that Wu et al. present TRIzol® as feasible means for combined extraction of proteins and RNA. Indeed, using TRI-reagent extraction for proteomics, they resolve two problems at a time: that of removing contaminating compounds and that of simultaneous analysis of gene and protein expression.

17ß-Estradiol induces changes in cytokine levels in head kidney and blood of juvenile sea bass (Dicentrarchus labrax, L., 1758).

The cytokine network is involved in the immune system communication. As estrogens influence the cytokine expression in mammals, this study investigated the impact of exogenous estrogenic pollutants on selected cytokines in Dicentrarchus labrax. The gene expression of Interleukin 6, Tumour Necrosis Factor α, Transforming Growth Factor ß1 and Interleukin 1ß was assessed and accomplished with protein measurements in the blood for the last two. Impacts through 17ß-estradiol mainly occurred at the beginning of organ regionalisation, thus falling together with a developmentally induced increase of Interleukin 1ß and Tumour Necrosis Factor α gene expression in 102 dph fish. 17ß-estradiol depressed this modification after 35 days of exposure and the cytokine gene expression tended to be generally down-regulated independently of the 17ß-estradiol concentrations after 56 days of exposure. This impact was confirmed at the protein level, showing that 17ß-estradiol affects the fine control of the cytokine network in sea bass.

Proteomic applications in ecotoxicology.

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.

Data processing and classification analysis of proteomic changes: a case study of oil pollution in the mussel, Mytilus edulis.

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.