Expression variations of two retinoid signaling pathway receptors in the rotifer Brachionus calyciflorus exposed to three endocrine disruptors.

Endocrine disruption compounds (EDC) are known to affect reproduction, development, and growth of exposed organisms. Although in vertebrates, EDCs mainly act through steroid receptors (e.g. androgen and estrogen receptors), their absence in many invertebrates suggests the involvement of another biological pathway in endocrine disruption effects. As retinoid signaling pathway is present in almost all Metazoa and its involvement in the endocrine disruption of gastropods (i.e. imposex) has been demonstrated, the present work was devoted to investigating the relative mRNA variations of two retinoid receptors genes, retinoid X receptor (RXR) and retinoid acid receptor (RAR), in the freshwater rotifer Brachionus calyciflorus exposed for 6, 12 and 24 h to flutamide, fenitrothion and cyproterone acetate, three anti-androgens known to disrupt sexual reproduction of Brachionus sp. Results revealed that fenitrothion did not affect the relative mRNA levels RXR and RAR in B. calyciflorus, whereas RXR and RAR mRNA levels could be significantly increased by 2 to 4.5-fold and from 2 to 7-fold after exposure to flutamide and cyproterone acetate, respectively. Moreover, the effects of flutamide and cyproterone acetate were measured from 6 and 12 h of exposure, respectively. Cyproterone acetate caused the highest increase of RXR and RAR mRNA levels, probably due to its progestin activity in addition to its anti-androgenic activity and the potential presence of a membrane-associated progesterone receptor as reported in Brachionus manjavacas. Consequently, although it is still difficult to evaluate the hormonal pathways involved in the endocrine disruption in Brachionus sp., this work suggests that the retinoid signaling pathway appears to be a good starting point to try to elucidate the molecular mechanisms involved in sexual reproductive dysfunction in Brachionidae.

Behavioural and biochemical alterations in gammarids as induced by chronic metallic exposures (Cd, Cu and Pb): Implications for freshwater biomonitoring.

In freshwater species, metal toxicity is usually assessed through short-term exposures, hence limiting the practical usefulness of biomarkers for monitoring long-term impacts on wildlife populations. This study investigates the biological alterations elicited by chronic metallic exposures in Gammarus fossarum using multi-level biomarkers. In aquaria, gammarids were exposed for 10 weeks to field-realistic concentrations of Cd, Cu or Pb (0.25, 1.5 or 5.0 µg/L). At the individual level, behavioural traits (respiration, locomotion and feeding) were compared between naive and chronically-exposed gammarids. At the cellular level, enzymatic activities involved in digestion, moult and cell stress were monitored after 2, 6 and 10 weeks of exposure in males and females to consider the temporal feature of their responses. Results showed that the inhibitory effects of Cd and Pb on respiration and locomotion disappeared in chronically-exposed gammarids, reflecting acclimation to maintain these processes, unlike Cu. Chronic Cu- and Pb-elicited feeding inhibition was associated with the inhibitions of digestion enzymes. Chitobiase was inhibited by Cu in males and, by Cd and Pb in females, suggesting gender-dependent disturbances in moulting. In both genders, Cd generated cellular stress by stimulating acidic phosphatase and peroxidase activities. To conclude, such cellular impairments and alterations in individual performances are likely to disturb individual growth, population dynamics and litter decomposition in the long-term. Besides, obtaining biological responses, common to metals or specific to a metal or a gender, supports the development of biomarkers highlighting long-term impacts of metals on the health of organisms and their associated ecological functions in natural environments.

Targeted impact of cyproterone acetate on the sexual reproduction of female rotifers.

Monogonont rotifers constitute, depending on the moment of the year, most of the zooplankton in many freshwater ecosystems. Sexual reproduction is essential in the development cycle of these organisms as it enables them to constitute stocks of cysts which can withstand adverse environmental conditions and hatch when favorable conditions return. However, endocrine disrupting compounds (EDCs) can interfere with the reproduction of organisms. The present work aimed to investigate the effects of cyproterone acetate (CPA, anti-androgen and progestogen synthetic steroid) at 0.5 mg L-1, on the sexual reproduction of Brachionus calyciflorus in a cross-mating experiment. Results show no impact on mixis whereas the fertilization rate and resting egg production were higher in females exposed to CPA (from embryogenesis to adult stage), regardless of the treatment applied to the males with which they were mating (i.e. males hatched from CPA-treated females or from control females). Moreover, neonate females which mothers has been exposed to 0.5 mg L-1 CPA had more oocytes in their germarium than control neonates. Our results suggest that the effects of CPA observed are not related to toxicity but rather are consistent with an endocrine disruption-related impact, probably through disturbance of the mate recognition protein (MRP) production and through interference with a steroid receptor. Moreover, the absence of effect on mixis rate indicates that mixis induction on the one hand and mating process and resting production on the other hand are not controlled by the same hormonal pathways.

Ecotoxicoproteomic assessment of the functional alterations caused by chronic metallic exposures in gammarids.

Very few ecotoxicological studies have been performed on long-term exposure under controlled conditions, hence limiting the assessment of the impact of chronic and diffuse chemical pressures on the health of aquatic organisms. In this study, an ecotoxicoproteomic approach was used to assess the integrated response and possible acclimation mechanisms in Gammarus fossarum following chronic exposures to Cd, Cu or Pb, at environmentally realistic concentrations (i.e. 0.25, 1.5 and 5 µg/L respectively). After 10-week exposure, changes in protein expression were investigated in caeca of control and exposed males. Gel-free proteomic analyses allowed for the identification of 35 proteins involved in various biological functions, for which 23 were significantly deregulated by metal exposures. The protein deregulation profiles were specific to each metal, providing evidence for metal-specific action sites and responses of gammarids. Among the tested metals, Cu was the most toxic in terms of mortality, probably linked with persistent oxidative stress. Moulting and osmoregulation were the major biological functions affected by Cu in the long-term. In Pb-exposed gammarids, significant deregulations of proteins involved in immune response and cytoskeleton were observed. Reproduction appears to be strongly affected in gammarids chronically exposed to Cd or Pb. Besides, modified expressions of several proteins involved in energy transfer and metabolism highlighted important energetic reshuffling to cope with chronic metal exposures. These results support the fact that metallic pressures induce a functional and energetic cost for individuals of G. fossarum with potential repercussions on population dynamics. Furthermore, this ecotoxicoproteomic study offers promising lines of enquiry in the development of new biomarkers that could make evidence of long-term impacts of metals on the health of organisms.

Corrigendum to "Transcriptome of the freshwater amphipod Gammarus pulex hepatopancreas" [Genomics Data 8 (2016) 91-92].

[This corrects the article DOI: 10.1016/j.gdata.2016.04.002.].

Transcriptome of the freshwater amphipod Gammarus pulex hepatopancreas.

So far, ecotoxicological studies used biomarkers of exposure or of effects in order to investigate the impacts of contaminated areas on biota (Peakall, 1994 [6]). However, although these results are important in the ecotoxicological risk assessment, biomarkers are very specific and only provide information on the biological processes or physiological pathways targeted by the biomarkers experimenters choose to test (Monsinjon and Knigge, 2007 [5]). In recent years, proteomics have become a major tool in ecotoxicology, as they provide a global insight into the mechanism of action of pollutants without the need of hypothesis testing or any preconception on the biological processes likely impacted (Gismondi et al., 2015; Trapp et al., 2015 [7]; Truebano, 2016 [8]). However, the analysis of proteomic results is often limited due to the lack of database, especially for non-model organisms, such as Gammarus sp, commonly used as biological model in ecotoxicology (Sornom et al., 2012 [11]; Vellinger et al., 2013 [9]; Gismondi and Thomé, 2014 [1]; Lebrun et al., 2014 [3]). Here, we performed Illumina HiSeq sequencing to total RNA isolated from the hepatopancreas (i.e. detoxification tissue) of Gammarus pulex males and females coming from uncontaminated river and contaminated river (e.g. PCB, benzo(a)pyrene). Approximately 290 M paired-end reads were assembled, filtered and sorted into 39,801 contigs whose 10.878 were similar of proteins available in databases. The assembled contigs could represent a reference hepatopancreas transcriptome for G. pulex, and constitute an important resource for future investigations on the impacts of pollutants on invertebrate biota, since it would improve the understanding of the mechanisms of action involved in toxicity. In addition, the hepatopancreas transcriptome will also allow the identification of new potential biomarkers for the ecotoxicological risk assessments. Assembled contigs were deposited in the European Nucleotide Archive under the BioProject number PRJEB13055, with accession numbers FJVI01000001-FJVI01039801.

Gender differences in responses in Gammarus pulex exposed to BDE-47: A gel-free proteomic approach.

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and females under control conditions (unexposed) and after 96h exposure to BDE-47. Using gel-free proteomic analysis, we have identified 45 proteins, of which 25 were significantly differently expressed according to sex and/or BDE-47 exposure. These proteins were involved in several biological processes such as energy metabolism, chaperone proteins, or transcription/translation. In unexposed amphipods, 11 proteins were significantly over-expressed in females, and 6 proteins were over-expressed in males. Under BDE-47 stress, 7 proteins were differently impacted according to sex. For example, catalase was over-expressed in exposed females and under-expressed in exposed males, as compared to respective controls. Conversely, proteins involved in energy metabolism were up-regulated in males and down-regulated in females. Our proteomic study showed differences in responses of males and females to BDE-47 exposure, emphasizing that sex is a confounding factor in ecotoxicological assessment. However, due to the limited information existing in databases on Gammarids, it was difficult to define a BDE-47 mechanism of action. The gel-free proteomic seems to be a promising method to develop in future ecotoxicological studies and thus, to improve our understanding of the mechanism of action of xenobiotics.

Behavioural and physiological responses of Gammarus fossarum (Crustacea Amphipoda) exposed to silver.

The study aims at investigating the effects of silver (Ag), a re-emerging contaminant, on physiological and behavioural responses in Gammarus fossarum. In a first experiment, G. fossarum Ag LC50s were evaluated during 96 h under semi-static mode of exposure. Juveniles appeared to be more sensitive to Ag (LC5096h: 1.01 µg L(-1)) than ovigerous females (LC5096h: 1.9 µg L(-1)) and adult males (LC5096h: 2.2 µg L(-1)). In a second experiment, the physiological (osmo-/ionoregulation; antioxidant enzymes; lipid peroxidation (LPO)) and behavioural (locomotor activity and ventilation) responses of male G. fossarum exposed to Ag (0, 0.5, 1, 2, and 4 µg L(-1)) were investigated. The mortality and Ag bioconcentration of gammarids exposed to Ag were significantly higher than controls. Concerning physiological responses, a 48 h-exposure to Ag had no impact on catalase activity but led to a significant decrease of haemolymph osmolality and [Na(+)]. On the contrary, LPO, Se-GPx and Na(+)/K(+)-ATPase activity were significantly increased. Behavioural responses, such as locomotor and ventilatory activities, were also significantly reduced in Ag exposed gammarids. After 96 h-exposure, especially to 0.5 µg Ag L(-1), most responses (ventilation, locomotor activity, haemolymph osmolality and [Na(+)]) were even more pronounced and haemolymph [Cl(-)] was significantly decreased but, contrary to observations after 48 h-exposure, Na(+)/K(+)-ATPase activity was significantly reduced. Our results demonstrate the drastic effects of realistic [Ag] concentration (0.5 µg Ag L(-1)) on an ubiquitous and functionally important freshwater invertebrate (implied in detritus breakdown), but also strongly suggest an energetic reallocation to the detriment of locomotor activity and in favour of maintenance functions (i.e., osmoregulation and detoxification). These results highlight the risk represented by Ag and the need to perform integrated studies (at different scales, from individual to ecosystem).

Do male and female gammarids defend themselves differently during chemical stress?

To investigate xenobiotic impacts on organism physiology, several studies involve biomarker assessment. However, most studies do not take into account the toxic effect on both males and females. Here, we have investigated the influence of gender on the detoxification response (reduced glutathione, metallothionein, γ-glutamylcystein ligase and carotenoid), energy reserves (protein, lipids and glycogen) and biomarker of toxic effects (malondialdehyde) in Gammarus roeseli exposed to cadmium. A principal component analysis revealed that G. roeseli males and females were differently impacted by cadmium. We observed lower malondialdehyde levels in females than in males, whatever the condition tested (i.e. control, 2 and 8 µg CdL(-1)), although the pattern of responses of control and exposures to 2 or 8 µgL(-1) was the same for both genders. Results could be linked to apparently more effective detoxification displayed by females than by males. Protein concentrations were unchanged in both genders, lipids contents were always significantly decreased and glycogen contents decreased only in females. This study supports the importance of taking into account the gender in ecotoxicological studies to have an overview of xenobiotics effects on a population.

Acanthocephalan parasites: help or burden in gammarid amphipods exposed to cadmium?

We investigated the influence of the acanthocephalan parasite Polymorphus minutus on the mortality of its intermediate host, Gammarus roeseli, exposed to cadmium, by the measure of LC(50-96h) values as well as the bioaccumulation of cadmium both in the host and in its parasite. LC(50) results revealed that infected G. roeseli males died less under cadmium stress than uninfected ones; while the converse has been observed in females. Cadmium resistance of infected males could be explained by a weaker bioconcentration factor (BCF) than in females. The lower BCF in infected individuals was closely related with an uptake of cadmium by P. minutus in its host. Nevertheless, although infected females had both weaker BCF and cadmium concentration in their body, the presence of P. minutus did not induce lower mortality than uninfected females. On the contrary, their sensitivity to cadmium was increased by the presence of P. minutus. We discuss the hypothesis that differences of mortality between uninfected and infected gammarids could be explained by a difference of cadmium bioconcentration in host, and by the cadmium bioaccumulation in the parasite. Indeed, results suggested that P. minutus could help G. roeseli to face with stress, what contributed to keep the host alive and favour the parasite transmission.

Does the acanthocephalan parasite Polymorphus minutus modify the energy reserves and antitoxic defences of its intermediate host Gammarus roeseli?

In disturbed environments, infected organisms have to face both parasitic and chemical stresses. Although this situation is common, few studies have been devoted to the effects of infection on hosts' energy reserves and antitoxic defence capacities, while parasite survival depends on host survival. In this study, we tested the consequences of an infection by Polymorphus minutus on the energy reserves (protein, lipid and glycogen) and antioxidant defence capacities (reduced glutathione, γ-glutamylcysteine ligase activity) of Gammarus roeseli males and females, in the absence of chemical stress. Moreover, malondialdehyde concentration was used as a toxicity biomarker. The results revealed that in infected G. roeseli, whatever their gender and the sampling month, protein and lipid contents were lower, but glycogen contents were higher. This could be explained by the fact that the parasite diverts part of the host's energy for its own development. Moreover, glutathione concentrations and γ-glutamylcysteine ligase activity were both lower, which could lead to lower antitoxic defence in the host. These results suggest negative effects on individuals in the case of additional stress (e.g. pollutant exposure). In the absence of chemical stress, the lower malondialdehyde level in infected gammarids could imply a probable protective effect of the parasite.