Effects of 17α-Ethinylestradiol (EE2) exposure during early life development on the gonadotropic axis ontogenesis of the European sea bass, Dicentrarchus labrax.

Exposure of young organisms to oestrogenic endocrine disrupting chemicals (EDCs) can elicit adverse effects, particularly on the reproductive function. In fish, as in other vertebrates, reproduction is controlled by the neuroendocrine gonadotropic axis, whose components are mainly regulated by sex steroids and may then be targets for EDCs. In the present study, we investigated the effects of a xenoestrogen exposure on the ontogenesis of the gonadotropic axis in European sea bass. After exposure of hatching larvae for 8 days to 17α-ethinylestradiol (EE2) (0.5 nM and 50 nM), gene expression for kisspeptins (kiss1, kiss2), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), gonadotropin beta subunits (lhß and fshß) and brain type aromatase (cyp19a1b) were measured using quantitative real-time PCR. Our results demonstrate that EE2 strongly stimulated the expression of brain type aromatase (cyp19a1b) in sea bass larvae. In addition, EE2 exposure also affected the mRNA levels of kiss1, gnrh1 and gnrh3 by inducing a downregulation of these genes during the early developmental stages, while no effect was seen in gnrh2, lhß and fshß. These results reinforce the idea that the larval development is a sensitive critical period in regard to endocrine disruption and that the gonadotropic axis in the developing sea bass is sensitive to xenoestrogen exposure.

The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors.

The marine mussel Mytilus edulis, tolerant to a wide range of environmental changes, combines a key role as a sentinel species for environmental monitoring programs and a significant economic importance. Mortality events caused by infective agents and parasites have not been described in mussels, which suggests an efficient immune system. This study aims at identifying the molecular mechanisms involved in the early immune responses M. edulis' hemocytes challenged with Vibrio splendidus LGP32 strain during 2, 4 and 6 h. A total of 149,296 assembled sequences has been annotated and compared to KEGG reference pathways. Several immune related sequences were identified such as Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy. Differential gene expression clustered 10 different groups of transcripts according to kinetics of transcript occurrence. Sequences were assigned to biological process gene ontology categories. Sequences encoding for galectins, fibrinogen-related proteins, TLRs, MyD88, some antimicrobial peptides, lysosomal hydrolases, heat shock proteins and protease inhibitors, as well as proteins of oxidative stress and apoptosis were identified as differently regulated during the exposure to V. splendidus LGP32. The levels of candidate transcripts were quantified in M. edulis' hemocytes exposed to V. splendidus LGP32 and 7SHRW by using branched DNA technology. Transcripts encoding for inhibitor kappa B, inhibitor of apoptosis proteins, tumor protein D54, serine/threonine-proteine kinase SIK2 were identified as up-regulated in hemocytes exposed to both strains.

Assessment of sperm quality in palaemonid prawns using Comet assay: methodological optimization.

The aim of this study was to adapt the Comet assay in spermatozoa of the marine prawn Palaemon serratus to use it as a marker of sperm quality. Indeed, due to the characteristics of their spermatozoa, the measurement of DNA integrity is one of the few markers which can be transferred to crustaceans to assess the quality of their semen. In the first step, the methods of collecting and maintaining spermatozoa were optimized. Cell survival was estimated during kinetics of preservation (i.e. 1, 2, 4 and 8 h) in various suspension media to define artificial seawater (ASW) as optimal. Several methods in the releasing of spermatozoa from the spermatophore of prawns were estimated with regard to their incidence both on the efficiency of extraction and the survival of cells. Pipetting up and down turned out to be the most successful and the least invasive technique. Secondly, the transfer of Comet assay was optimized by studying various times in both cell lysis (i.e. 1, 6, 18 h) and DNA denaturation (i.e. 15, 30 and 45 min), after in vitro exposure of spermatozoa to an H2O2 gradient as model genotoxicant. Results revealed that a minimum of 1 h in cell lysis and 15 min of DNA denaturation were sufficient to obtain valuable results, linked with a low compaction of DNA in spermatozoa of Palaemon sp. Finally, the sensitivity of P. serratus spermatozoa was assessed after in vitro exposures to model genotoxicants displaying various modes of interaction with DNA (i.e. UV-C, 13.3-79.5 J m-2; H2O2, 5-10 µM and MMS, 0.5-5 mM) and some environmental contaminants known or suspected to be genotoxic (i.e. cadmium and diuron, 0.015-1.5 µg L-1; carbamazepine, 0.1-10 µg L-1) for invertebrates. The low variability of the baseline level of DNA strand breaks recorded in controls highlighted the robustness of the method. P. serratus spermatozoa displayed significant DNA damage from the lowest doses tested for all model genotoxicants, but conversely, no genotoxic effect of tested environmental contaminants was observed. These results, which are discussed according to the protocol tested in the present study and the comparison with literature data, could suggest a difference in the response or sensitivity of spermatozoa to environmental genotoxicity between invertebrate species, and therefore the interest of Palaemonidae prawns in ecogenotoxicology. In conclusion, the present study underlines the potential of the Comet assay as a marker to assess the contamination impact on the sperm quality in Palaemonidae prawns in view to a potential application for in situ biomonitoring surveys.

Transcriptome analysis of the copepod Eurytemora affinis upon exposure to endocrine disruptor pesticides: Focus on reproduction and development.

Copepods-which include freshwater and marine species-represent the most abundant group of aquatic invertebrates. Among them, the calanoid copepod Eurytemora affinis is widely represented in the northern hemisphere estuaries and has become a species of interest in ecotoxicology. Like other non-target organisms, E. affinis may be exposed to a wide range of chemicals such as endocrine disruptors (EDs). This study investigated the gene expression variation in E. affinis after exposure to ED pesticides-chosen as model EDs-in order to (i) improve the knowledge on their effects in crustaceans, and (ii) highlight relevant transcripts for further development of potential biomarkers of ED exposure/effect. The study focused on the reproduction function in response to ED. Copepods were exposed to sublethal concentrations of pyriproxyfen (PXF) and chlordecone (CLD) separately. After 48h, males and females (400 individuals each) were sorted for RNA extraction. Their transcriptome was pyrosequenced using the Illumina(®) technology. Contigs were blasted and functionally annotated using Blast2GO(®). The differential expression analysis between ED- and acetone-exposed organisms was performed according to sexes and contaminants. Half of the 19,721 contigs provided by pyrosequencing were annotated, mostly (80%) from arthropod sequences. Overall, 2,566 different genes were differentially expressed after ED exposures in comparison with controls. As many genes were differentially expressed after PXF exposure as after CLD exposure. In contrast, more genes were differentially expressed in males than in females after both exposures. Ninety-seven genes overlapped in all conditions. Finally, 31 transcripts involved in reproduction, growth and development, and changed in both chemical exposures were selected as potential candidates for future development of biomarkers.

Functional and molecular responses in Mytilus edulis hemocytes exposed to bacteria, Vibrio splendidus.

This study aims at examining the morphological, functional and molecular responses of Mytilus edulis hemocytes exposed to different strains of Gram-negative bacteria Vibrio splendidus (a virulent strain V. splendidus LGP32, V. splendidus LGP32 Δvsm without metalloprotease and an environmental type strain V. splendidus 7SHRW) at a 1:3 ratio for 2, 4, and 6 h. Our data showed that hemocytes could have a discriminative capacity towards microorganisms. Both V. splendidus LGP32 strains had an effect on hemocyte adhesion, phagocytosis abilities and oxidative burst, whereas the environmental strain 7SHRW induced weak and delayed hemocyte responses. At a molecular level, differential levels of candidate transcripts were measured in M. edulis hemocytes exposed to V. splendidus LGP32-GFP and 7SHRW. Mainly, a down-regulation of defensin was recorded in hemocytes exposed to V. splendidus LGP32. A significant up-regulation of lysozyme and proteasome 26S was observed at 2 h followed by a down-regulation at 4 and 6 h of exposure to the LGP32 strain. Similarly, SOD and GPx genes were up-regulated 2 h post-exposure to LGP32 strain and their expressions decreased after 4 and 6 h post-exposure. Further analysis is however needed in a near future to relate the transcript level variations with the physiological process.

Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain.

In the past decades, reports on bivalves' pathogens and associated mortalities have steadily increased. To face pathogenic micro-organisms, bivalves rely on innate defenses established in hemocytes which are essentially based on phagocytosis and cytotoxic reactions. As a step towards a better understanding of the molecular mechanisms involved in the mussel Mytilus edulis innate immune system, we constructed and sequenced a normalized cDNA library specific to M. edulis hemocytes unchallenged (control) and challenged with Vibrio splendidus LGP32 strain for 2, 4 and 6 h. A total of 1,024,708 nucleotide reads have been generated using 454 pyrosequencing. These reads have been assembled and annotated into 19,622 sequences which we believe cover most of the M. edulis hemocytes transcriptome. These sequences were successfully assigned to biological process, cellular component, and molecular function Gene Ontology (GO) categories. Several transcripts related to immunity and stress such as some fibrinogen related proteins and Toll-like receptors, the complement C1qDC, some antioxidant enzymes and antimicrobial peptides have already been identified. In addition, Toll-like receptors signaling pathways and the lysosome and apoptosis mechanisms were compared to KEGG reference pathways. As an attempt for large scale RNA sequencing, this study focuses on identifying and annotating transcripts from M. edulis hemocytes regulated during an in vitro experimental challenge with V. splendidus. The bioinformatic analysis provided a reference transcriptome, which could be used in studies aiming to quantify the level of transcripts using high-throughput analysis such as RNA-Seq.

Transcriptome analysis of neoplastic hemocytes in soft-shell clams Mya arenaria: Focus on cell cycle molecular mechanism.

In North America, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). Disseminated neoplasia is commonly recognized as a tetraploid disorder related to a disruption of the cell cycle. However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts related to DN in soft shell clams' hemocytes using next generation of sequencing (Illumina HiSeq2000). This study mainly focuses on transcripts and molecular mechanisms involved in cell cycle. Using Illumina next generation of sequencing, more than 95,399,159 reads count with an average length of 45 bp was generated from three groups of hemocytes: (1) a healthy group with less than 10% of tetraploid cells; (2) an intermediate group with tetraploid hemocytes ranging between 10% and 50% and (3) a diseased group with more than 50% of tetraploid cells. After the reads were cleaned by removing the adapters, de novo assembly was performed on the sequences and more than 73,696 contigs were generated with a mean contig length estimated at 585 bp ranging from 189 bp to 14,773 bp. Once a Blastx search against NCBI Non Redundant database was performed and the duplicates removed, 18,378 annotated sequences matched known sequences, 3078 were hypothetical and 9002 were uncharacterized sequences. Fifty percent and 41% of known sequences match sequences from Mollusca and Gastropoda respectively. Among the bivalvia, 33%, 17%, 17% and 15% of the contigs match sequences from Ostreoida, Veneroida, Pectinoida and Mytiloida respectively. Gene ontology analysis showed that metabolic, cellular, transport, cell communication and cell cycle represent 33%, 15%, 9%, 8.5% and 7% respectively of the total biological process. Approximately 70% of the component process is related to intracellular process and 15% is linked to protein and ribonucleoprotein complex. Catalytic activities and binding molecular processes represent 39% and 33% of the total molecular functions. Interestingly, nucleic acid binding represents more than 18% of the total protein class. Transcripts involved in the molecular mechanisms of cell cycle are discussed providing new avenues for future investigations.

Molecular characterization and mRNA expression of grp78 and hsp90A in the estuarine copepod Eurytemora affinis.

The present study aimed to develop a method of quantification of heat shock protein transcript levels in the estuarine copepod Eurytemora affinis. For that, the full-length cDNA of the 78-kDa glucose-regulated protein (Ea-grp78) and the cytosolic 90-kDa heat shock protein (Ea-hsp90A) from this species have been cloned. These cDNA revealed, respectively, 2,370 and 2,299 bp with 1,971 and 2,124 bp open reading frames encoding 656 and 707 amino acids. Main features, sequence identities and phylogenetic analysis with other species were described. Then, the expression profiles were analysed using reverse transcription/real-time quantitative PCR method from copepods subjected to different thermic and osmotic stresses in laboratory, and from copepods directly sampled into the natural population of the Seine Estuary (France) along a salinity gradient. Thermic shock (7.5°C, 22.5°C and 30°C during 90 min) significantly induced increases of transcript quantities ranged between 1.7- and 19.7-fold the levels observed in control conditions (15°C). Hypo- and hyper-osmotic shocks (salinities of 1 and 30 during 90 min) caused a 2-fold induction of Ea-hsp90A transcript level in comparison to controls (salinity of 15) whereas no significant change was measured for Ea-grp78. On the other hand, similar expression profiles were observed for the two transcripts after 72 h of exposition to salinities of 1 and 25 with a significant 2-fold induction observed for the lower salinity. To finish, strong expression inductions of both Ea-grp78 and Ea-hsp90A genes were observed in field copepods sampled at low salinity during the campaigns of June 2009 and May 2010. These results tend to show that the low salinity and the increase of temperature seem to have a synergic effect on stress condition of copepods.

Induction of transposase and polyprotein RNA levels in disseminated neoplastic hemocytes of soft-shell clams: Mya arenaria.

In Prince Edward Island, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts involved in the development of the disease. Four subtractive cDNA sequence libraries were generated and more than 200,000 reads were obtained. Following similarity searches in genome databases, the transcripts were assigned to cellular functions including mitochondrial respiration, structural proteins, cytoskeleton, nucleic acid regulation, general metabolism, signal transduction, apoptosis, cell cycle regulation, as well as virus transcripts. The expression levels of transposase and polyprotein genes were evaluated in clams with various percentages of tetraploid hemocytes. Data have shown that expression levels were significantly higher in clams with a high percentage of tetraploid hemocytes. These results reinforce the hypothesis of endogenous retrotransposon involvement in the etiology of the disease. Further investigations are needed, however, to elucidate the role of transposase and polyprotein in the disease development.

Molecular cloning of flounder Xp18, a newly identified highly conserved protein mainly expressed in the ovary.

Screening of a flounder ovary cDNA library with a rainbow trout p53 probe led to the isolation of a p53-unrelated cDNA encoding an unknown 161 amino acid protein. In view of its apparent molecular weight and yet unknown function, the deduced protein was named Xp18. Corresponding orthologous cDNAs or expressed sequence tags have been identified in several species including human, rodents, bovine, chicken and zebrafish and a related cDNA has also been isolated in the fruit fly. Deduced amino acid sequences appeared to be extremely well conserved throughout vertebrate evolution. Structure predictions suggested that Xp18 may correspond to an integral protein comprising four transmembrane domains. The charged C-termini of all known vertebrate Xp18-like proteins displayed a characteristic KXKXX motif which is considered as an endoplasmic reticulum targeting sequence. Gene expression, as shown by Northern blot and quantitative reverse transcription-polymerase chain reaction analysis, was significantly higher in the ovary and to a lesser extent in the brain. Xp18 transcripts were also detected by in situ hybridization in most of the circumventricular regions of the brain of adult flounders. The gene encoding the human protein is located on chromosome Xq22.1, a genome region involved in numerous genetic diseases including premature ovarian failure.