Impact of environmental micropollutants and diet composition on the gut microbiota of wild european eels (Anguilla anguilla).

In fish, the gut microbiome plays a crucial role in homeostasis and health and is affected by several organic and inorganic environmental contaminants. Amphidromous fish are sentinel species, particularly exposed to these stressors. We used whole metagenome sequencing to characterize the gut microbiome of wild European eels (Anguilla anguilla) at a juvenile stage captured from three sites with contrasted pollution levels in term of heavy metals and persistent organic pollutants. The objectives were to identify what parameters could alter the gut microbiome of this catadromous fish and to explore the potential use of microbiota as bioindicators of environment quality. We identified a total of 1079 microbial genera. Overall, gut microbiome was dominated by Proteobacteria, Firmicutes and Actinobacteria. Alpha and beta diversity were different amongst sites and could be explained by a reduced number of environmental and biological factors, specifically the relative abundance of fish preys in eels' diet, PCB101, γHCH (lindane), transnonachlor and arsenic. Furthermore, we identified a series of indicator taxa with differential abundance between the three sites. Changes in the microbial communities in the gut caused by environmental pollutants were previously undocumented in European eels. Our results indicate that microbiota might represent another route by which pollutants affect the health of these aquatic sentinel organisms.

The underestimated toxic effects of nanoplastics coming from marine sources: A demonstration on oysters (Isognomon alatus).

This study aims to assess the potential toxicity of (1) nanoplastics (NPs) issued from the fragmentation of larger plastic particles collected on the Caribbean marine coast (NP-G), and (2) polystyrene NPs (NP-PS), commonly used in the literature, on Caribbean swamp oysters (Isognomon alatus). Oysters were exposed to 7.5 and 15 µg.L-1 of each type of NPs, combined or not with arsenic (As) at 1 mg.L-1 for one week before molecular analyses at gene levels. Overall, the NP-G triggered more significant changes than NP-PS, especially when combined with As. Genes involved in the mitochondrial metabolism were strongly up-regulated in most of the conditions tested (up to 11.6 fold change for the NP-PS exposure at 7.5 µg.L-1 for the 12s). NPs in combination with As or not triggered a response against oxidative stress, with an intense repression of cat and sod1 (0.01 fold-changes for the NP-G condition at 7.5 µg.L-1). Both NP-G and NP-PS combined or not with As led to an up-regulation of apoptotic genes p53 and bax (up to 59.3 fold-changes for bax in the NP-G condition with As). Our study reported very innovative molecular results on oysters exposed to NPs from environmental sources. Our results suggest that the composition, surface charge, size, and the adsorbed contaminants of plastics from the natural environment may have synergic effects with plastic, which are underestimated when using manufactured NPs as NP-PS in ecotoxicological studies.

Transfer and Transcriptomic Profiling in Liver and Brain of European Eels (Anguilla anguilla) After Diet-borne Exposure to Gold Nanoparticles.

A nanometric revolution is underway, promising technical innovations in a wide range of applications and leading to a potential boost in environmental discharges. The propensity of nanoparticles (NPs) to be transferred throughout trophic chains and to generate toxicity was mainly assessed in primary consumers, whereas a lack of knowledge for higher trophic levels persists. The present study focused on a predatory fish, the European eel (Anguilla anguilla) exposed to gold NPs (AuNPs; 10 nm, polyethylene glycol-coated) for 21 d at 3 concentration levels in food: 0 (NP0), 1 (NP1), and 10 (NP10) mg Au kg-1 . Transfer was assessed by Au quantification in eel tissues, and transcriptomic responses in the liver and brain were revealed by a high-throughput RNA-sequencing approach. Eels fed at NP10 presented an erratic feeding behavior, whereas Au quantification only indicated transfer to intestine and kidney of NP1-exposed eels. Sequencing of RNA was performed in NP0 and NP1 eels. A total of 258 genes and 156 genes were significantly differentially transcribed in response to AuNP trophic exposure in the liver and brain, respectively. Enrichment analysis highlighted modifications in the immune system-related processes in the liver. In addition, results pointed out a shared response of both organs regarding 13 genes, most of them being involved in immune functions. This finding may shed light on the mode of action and toxicity of AuNPs in fish. Environ Toxicol Chem 2020;39:2450-2461. © 2020 SETAC.

Identification and expression of microRNAs in european eels Anguilla anguilla from two natural sites with different pollution levels.

MicroRNAs (miRNAs) are a class of small non-coding RNA that control multiple biological processes through negative post-transcriptional regulation of gene expression. Recently a role of miRNAs in the response of aquatic organisms to environmental toxicants emerged. Toxicant-induced changes in miRNA expression might then represent novel biomarkers to evaluate the health status of these organisms. In this study, we aimed to identify the miRNA repertoire in the liver of the European eel Anguilla anguilla and to compare their differential expression between a polluted site located in the Gironde Estuary and a pristine site in Arcachon Bay (France). A total of 299 mature miRNAs were identified. In polluted water, 19 miRNAs were up-regulated and 22 were down-regulated. We predicted that these differentially expressed miRNAs could target 490 genes that were involved in ribosome biogenesis, response to hormones, response to chemical and chromatin modification. Moreover, we observed only few examples (29) of negative correlation between the expression levels of miRNAs and their targets suggesting that, in the system studied, miRNAs might not only regulate gene expression directly by degrading mRNA but also by inhibiting protein translation or by regulating other epigenetic processes. This study is the first example of in situ investigation of the role of miRNAs in the response of a fish species to water quality. Our findings provide new insights into the involvement of epigenetic mechanisms in the response of animals chronically exposed to pollution and pave the way for the utilization of miRNAs in aquatic ecotoxicology.

Whole-transcriptome response to wastewater treatment plant and stormwater effluents in the Asian clam, Corbicula fluminea.

The increase in human population and urbanization are resulting in an increase in the volume of wastewater and urban runoff effluents entering natural ecosystems. These effluents may contain multiple pollutants to which the biological response of aquatic organisms is still poorly understood mainly due to mixture toxicity and interactions with other environmental factors. In this context, RNA sequencing was used to assess the impact of a chronic exposure to wastewater treatment plant and stormwater effluents at the whole-transcriptome level and evaluate the potential physiological outcomes in the Asian clam Corbicula fluminea. We de-novo assembled a transcriptome from C. fluminea digestive gland and identified a set of 3,181 transcripts with altered abundance in response to water quality. The largest differences in transcriptomic profiles were observed between C. fluminea from the reference site and those exposed to wastewater treatment plant effluents. On both anthropogenically impacted sites, most differentially expressed transcripts were involved in signaling pathways in relation to energy metabolism such as mTOR and FoxO, suggesting an energy/nutrient deficit and hypoxic conditions. These conditions were likely responsible for damages to proteins and transcripts in response to wastewater treatment effluents whereas exposure to urban runoff might result in immune and endocrine disruptions. In absence of comprehensive chemical characterization, the RNAseq approach could provide information regarding the mode of action of pollutants and then be useful for the identification of which parameters must be studied at higher integration level in order to diagnose sites where the presence of complex and variable mixtures of chemicals is suspected.

Transcriptomic responses of the endangered freshwater mussel Margaritifera margaritifera to trace metal contamination in the Dronne River, France.

The freshwater pearl mussel Margaritifera margaritifera is one of the most threatened freshwater bivalves worldwide. In this study, we aimed (i) to study the processes by which water quality might affect freshwater mussels in situ and (ii) to provide insights into the ecotoxicological significance of water pollution to natural populations in order to provide necessary information to enhance conservation strategies. M. margaritifera specimens were sampled in two close sites located upstream or downstream from an illegal dumping site. The renal transcriptome of these animals was assembled and gene transcription determined by RNA-seq. Correlations between transcription levels of each single transcript and the bioaccumulation of nine trace metals, age (estimated by sclerochronology), and condition index were determined in order to identify genes likely to respond to a specific factor. Amongst the studied metals, Cr, Zn, Cd, and Ni were the main factors correlated with transcription levels, with effects on translation, apoptosis, immune response, response to stimulus, and transport pathways. However, the main factor explaining changes in gene transcription appeared to be the age of individuals with a negative correlation with the transcription of retrotransposon-related genes. To investigate this effect further, mussels were classified into three age classes. In young, middle-aged and old animals, transcription levels were mainly explained by Cu, Zn and age, respectively. This suggests differences in the molecular responses of this species to metals during its lifetime that must be better assessed in future ecotoxicology studies.

Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification.

The bicarbonate ion (HCO3(-)) is involved in two major physiological processes in corals, biomineralization and photosynthesis, yet no molecular data on bicarbonate transporters are available. Here, we characterized plasma membrane-type HCO3(-) transporters in the scleractinian coral Stylophora pistillata. Eight solute carrier (SLC) genes were found in the genome: five homologs of mammalian-type SLC4 family members, and three of mammalian-type SLC26 family members. Using relative expression analysis and immunostaining, we analyzed the cellular distribution of these transporters and conducted phylogenetic analyses to determine the extent of conservation among cnidarian model organisms. Our data suggest that the SLC4γ isoform is specific to scleractinian corals and responsible for supplying HCO3(-) to the site of calcification. Taken together, SLC4γ appears to be one of the key genes for skeleton building in corals, which bears profound implications for our understanding of coral biomineralization and the evolution of scleractinian corals within cnidarians.

Transcriptome analysis of the scleractinian coral Stylophora pistillata.

The principal architects of coral reefs are the scleractinian corals; these species are divided in two major clades referred to as "robust" and "complex" corals. Although the molecular diversity of the "complex" clade has received considerable attention, with several expressed sequence tag (EST) libraries and a complete genome sequence having been constructed, the "robust" corals have received far less attention, despite the fact that robust corals have been prominent focal points for ecological and physiological studies. Filling this gap affords important opportunities to extend these studies and to improve our understanding of the differences between the two major clades. Here, we present an EST library from Stylophora pistillata (Esper 1797) and systematically analyze the assembled transcripts compared to putative homologs from the complete proteomes of six well-characterized metazoans: Nematostella vectensis, Hydra magnipapillata, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Ciona intestinalis and Homo sapiens. Furthermore, comparative analyses of the Stylophora pistillata ESTs were performed against several Cnidaria from the Scleractinia, Actiniaria and Hydrozoa, as well as against other stony corals separately. Functional characterization of S. pistillata transcripts into KOG/COG categories and further description of Wnt and bone morphogenetic protein (BMP) signaling pathways showed that the assembled EST library provides sufficient data and coverage. These features of this new library suggest considerable opportunities for extending our understanding of the molecular and physiological behavior of "robust" corals.

Carbonic anhydrases in anthozoan corals-A review.

Coral reefs are among the most biologically diverse and economically important ecosystems on the planet. The deposition of massive calcium carbonate skeletons (biomineralization or calcification) by scleractinian corals forms the coral reef framework/architecture that serves as habitat for a large diversity of organisms. This process would not be possible without the intimate symbiosis between corals and photosynthetic dinoflagellates, commonly called zooxanthellae. Carbonic anhydrases play major roles in those two essential processes of coral's physiology: they are involved in the carbon supply for calcium carbonate precipitation as well as in carbon-concentrating mechanisms for symbiont photosynthesis. Here, we review the current understanding of diversity and function of carbonic anhydrases in corals and discuss the perspective of theses enzymes as a key to understanding impacts of environmental changes on coral reefs.

A new coral carbonic anhydrase in Stylophora pistillata.

Scleractinian corals are of particular interest due to their ability to establish an intracellular mutualistic symbiosis with phototrophic dinoflagellates and to deposit high rates of calcium carbonate in their skeleton. Carbonic anhydrases have been shown to play a crucial role in both processes. In this study, we report the molecular cloning and characterization of a novel α-CA in the coral Stylophora pistillata. This enzyme shares homologies with the human isoform CA II and is referred to as STPCA-2. STPCA-2 is 35.2 kDa and possesses all key amino acids for catalytic activity. With a ratio between catalytic and Michaelis constants (k(cat)/K(m)) of 8.3.10(7) M(-1) s(-1) is considered as highly active. Owing to its intracellular localisation in the oral endoderm and in the aboral tissue, we propose that STPCA-2 is involved in pH regulation and/or inorganic carbon delivery to symbiont and calcification.

Carbonic anhydrase inhibitors. Inhibition studies with anions and sulfonamides of a new cytosolic enzyme from the scleractinian coral Stylophora pistillata.

The catalytic activity and the inhibition of a new coral carbonic anhydrase (CA, EC 4.2.1.1), from the scleractinian coral Stylophora pistillata, STPCA-2, has been investigated. STPCA-2 has high catalytic activity for the physiological reaction being less sensitive to anion and sulfonamide inhibitors compared to STPCA, a coral enzyme previously described. The best STPCA-2 anion inhibitors were sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (K(I)s of 5.7-67.2µM) whereas the best sulfonamide inhibitors were acetazolamide and dichlorophenamide (K(I)s of 74-79nM). Because this discriminatory effect between these two coral CAs, sulfonamides may be useful to better understand the physiological role of STPCA and STPCA-2 in corals and biomineralization processes.

Carbonic anhydrase activators. The first activation study of a coral secretory isoform with amino acids and amines.

The activity of the coral Stylophora pystillata secretory carbonic anhydrase STPCA has been tested in presence of amino acids and amines. All the investigated compounds showed a positive, activating effect on k(cat) and have been separated in weak (K(A) in the range of 21-126 microM), medium (10.1-19 microM) and strong enzyme activators (K(A) of 0.18-3.21 microM). D-DOPA was found to be the best coral enzyme activator, with an activation constant K(A) of 0.18 microM. This enhancement of STPCA activity, as well as previous enzyme inhibition results, might now be tested on living organisms to better understand the role played by these enzymes in the coral calcification processes.

Symbiosis-dependent gene expression in coral-dinoflagellate association: cloning and characterization of a P-type H+-ATPase gene.

We report the molecular cloning of a H(+)-ATPase in the symbiotic dinoflagellate, Symbiodinium sp. previously suggested by pharmacological studies to be involved in carbon-concentrating mechanism used by zooxanthellae when they are in symbiosis with corals. This gene encodes a protein of 975 amino acids with a calculated mass of about 105 kDa. The structure of the protein shows a typical P-type H(+)-ATPase structure (type IIIa plasma membrane H(+)-ATPases) and phylogenetic analyses show that this new proton pump groups with diatoms in the Chromoalveolates group. This Symbiodinium H(+)-ATPase is specifically expressed when zooxanthellae are engaged in a symbiotic relationship with the coral partner but not in free-living dinoflagellates. This proton pump, therefore, could be involved in the acidification of the perisymbiotic space leading to bicarbonate dehydration by carbonic anhydrase activity in order to supply inorganic carbon for photosynthesis as suggested by earlier studies. To our knowledge, this work provides the first example of a symbiosis-dependent gene in zooxanthellae and confirms the importance of H(+)-ATPase in coral-dinoflagellate symbiosis.

Carbonic anhydrase inhibitors. Inhibition studies of a coral secretory isoform by sulfonamides.

The inhibition of a newly cloned coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug), as well as the sulfamate antiepileptic topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with low potency inhibitors (K(I)s in the range of 163-770nM), or with medium potency inhibitors (K(I)s in the range of 75.1-105nM), whereas ethoxzolamide, several clinically used sulfonamides and heterocyclic compounds showed stronger potency, with K(I)s in the range of 16-48.2nM. These inhibitors may be useful to better understand the physiological role of the Stylophora pistillata CA (STPCA) in corals and its involvement in biomineralisation in this era of global warming.

Carbonic anhydrase inhibitors: inhibition studies of a coral secretory isoform with inorganic anions.

The inhibition of a coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate. The full-length scleractinian coral Stylophora pistillata CA, STPCA, has a significant catalytic activity for the physiological reaction of CO(2) hydration to bicarbonate, similarly to the ubiquitous human isoforms hCA I (cytosolic) and hCA VI (secreted). The best STPCA anion inhibitors were bromide, iodide, carbonate, and sulfamate, with inhibition constants of 9.0-10.0 microM.

Carbonic anhydrase in the scleractinian coral Stylophora pistillata: characterization, localization, and role in biomineralization.

Carbonic anhydrases (CA) play an important role in biomineralization from invertebrates to vertebrates. Previous experiments have investigated the role of CA in coral calcification, mainly by pharmacological approaches. This study reports the molecular cloning, sequencing, and immunolocalization of a CA isolated from the scleractinian coral Stylophora pistillata, named STPCA. Results show that STPCA is a secreted form of alpha-CA, which possesses a CA catalytic function, similar to the secreted human CAVI. We localized this enzyme at the calicoblastic ectoderm level, which is responsible for the precipitation of the skeleton. This localization supports the role of STPCA in the calcification process. In symbiotic scleractinian corals, calcification is stimulated by light, a phenomenon called "light-enhanced calcification" (LEC). The mechanism by which symbiont photosynthesis stimulates calcification is still enigmatic. We tested the hypothesis that coral genes are differentially expressed under light and dark conditions. By real-time PCR, we investigated the differential expression of STPCA to determine its role in the LEC phenomenon. Results show that the STPCA gene is expressed 2-fold more during the dark than the light. We suggest that in the dark, up-regulation of the STPCA gene represents a mechanism to cope with night acidosis.