Stress-related gene transcription in fish exposed to parasitic larvae of two freshwater mussels with divergent infection strategies.

Freshwater unionoid mussels have a unique life cycle involving a temporary parasitic phase. Their larvae (glochidia) attach to the gills or fins of fish hosts where they remain encysted until metamorphosis into free-living juveniles. The physiological response of fish during the critical period of glochidial attachment is not well understood, but recent work suggests that glochidia retention and survival is enhanced in stressed and cortisol-injected hosts. In this study, the early changes induced by glochidiosis were investigated for the first time at the transcriptional level. In 2 separate experiments, juvenile yellow perch Perca flavescens were inoculated with glochidia of Elliptio complanata (a host generalist) and Lampsilis radiata (a host specialist) following a standardized procedure. The transcriptional levels of 5 genes involved in the fish response to stress were assessed in the host liver and gills 24 h post-infection using quantitative real-time PCR. The number of encysted glochidia did not significantly differ between fish inoculated with E. complanata and L. radiata. Both species induced a 3-fold increase of 70 kDa heat-shock protein gene (hsp70) transcription in host liver. However, only E. complanata influenced the transcription of cortisol-regulated genes, notably glucocorticoid receptor DNA-binding factor 1 (grlf1). This gene, known to modulate tissue responsiveness to cortisol, was downregulated in infected fish compared to controls. Our findings suggest that different glochidia species interact with their fish host in distinct ways. Additional studies are required to address this hypothesis and further investigate the significance of the observed host transcriptional responses.

RNA-sequencing to assess the health of wild yellow perch (Perca flavescens) populations from the St. Lawrence River, Canada.

This study aimed to better understand in situ cumulative effects of anthropogenic stressors on the health of St. Lawrence River (QC, Canada) yellow perch populations using high-throughput transcriptomics and a multi-biological level approach. Fish were collected in the upstream fluvial Lake Saint-François (LSF) with low degree of environmental perturbations; Lake Saint-Louis (LSL) considered having a moderate degree of anthropogenic stressors, and Lake Saint-Pierre (LSP) a sector where the perch population has been severely declining. Morphometric results indicated that fish from the downstream LSP showed lower body condition compared to LSF and LSL. Liver transcriptomic responses were assessed by RNA-sequencing. Two hundred and eighty genes were over-transcribed in LSP perch while 200 genes were under-transcribed compared to LSF and LSL. In LSP fish, genes transcripts related to reproduction, retinol, iron, thyroid hormones, oxidative stress, lipid metabolism and immune functions were among the most abundant suggesting that multiple metabolic and physiological pathways were impacted by environmental stressors at this site. Inhibition of liver superoxide dismutase, catalase and glutathione S-transferase activities were also observed at the cellular level. Overall, identified impacted biological pathways in perch from LSP may help understand the precarious state of this population and identify the factors inhibiting its recovery.

Multigenerational effects evaluation of the flame retardant tris(2-butoxyethyl) phosphate (TBOEP) using Daphnia magna.

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphate ester used as substitute following the phase-out of brominated flamed retardants. Because of its high production volume and its use in a broad range of applications, this chemical is now frequently detected in the environment and biota. However, limited information is available on the long-term effects of TBOEP in aquatic organisms. In this study, Daphnia magna were exposed over three 21d generations to an environmentally relevant concentration of TBOEP (10µg/L) and effects were evaluated at the gene transcription, protein, and life-history (i.e., survival, reproduction and growth) levels. Chronic exposure to TBEOP did not impact survival or reproduction of D. magna but affected the growth output. The mean number of molts was also found to be lower in daphnids exposed to the chemical compared to control for a given generation, however there were no significant differences over the three generations. Molecular responses indicated significant differences in the transcription of genes related to growth, molting, ecdysteroid and juvenile hormone signaling, proteolysis, oxidative stress, and oxygen transport within generations. Levels of mRNA were also found to be significantly different for genes known to be involved in endocrine-mediated mechanisms such as reproduction and growth between generations F0, F1, and F2, indicating effects of parental exposure on offspring. Transcription results were supported by protein analyses with the significant decreased in catalase (CAT) activity in F1 generation, following the decreased transcription of cat in the parental generation. Taken together, these multi-biological level results suggest long-term potential endocrine disruption effects of TBOEP in D. magna exposed to an environmentally relevant concentration. This study highlights the importance of using chronic and multigenerational biological evaluation to assess risks of emerging chemicals.

Effects of food-borne exposure of juvenile rainbow trout (Oncorhynchus mykiss) to emerging brominated flame retardants 1,2-bis(2,4,6-tribromophenoxy)ethane and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate.

Brominated flame retardants (BFRs) represent a large group of chemicals used in a variety of household and commercial products to prevent fire propagation. The environmental persistence and toxicity of some of the most widely used BFRs has resulted in a progressive ban worldwide and the development of novel BFRs for which the knowledge on environmental health impacts remains limited. The objectives of this study were to evaluate the effects of two emerging BFRs, 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB), in diet exposed juvenile rainbow trout (Oncorhynchus mykiss). Both compounds were detected in fish carcasses at 76% and 2% of the daily dosage of BTBPE and EH-TBB, respectively, indicating accumulation of BTBPE and by contrast extensive depuration/metabolism of EH-TBB. Liver gene transcription analysis using RNA-sequencing indicated that the chronic 28-d dietary exposure of trout to EH-TBB down-regulated one single gene related to endocrine-mediated processes, whereas BTBPE impacted the transcription of 33 genes, including genes involved in the immune response, reproduction, and oxidative stress. Additional analysis using qRT-PCR after 48-h and 28-d of exposure confirmed the impact of BTBPE on immune related genes in the liver (apolipoprotein A-I, lysozyme) and the head-kidney (complement c3-4). However, the activity of lysozymes measured at the protein level did not reflect transcriptomic results. Overall, results suggested an impact on immune-related gene transcription in BTBPE exposed fish, as well as oxidative stress and endocrine disruption potentials.

Transcriptomic, cellular and life-history responses of Daphnia magna chronically exposed to benzotriazoles: Endocrine-disrupting potential and molting effects.

Benzotriazoles (BZTs) are ubiquitous aquatic contaminants used in a wide range of industrial and domestic applications from aircraft deicers to dishwasher tablets. Acute toxicity has been reported in aquatic organisms for some of the BZTs but their mode of action remains unknown. The objectives of this study were to evaluate the transcriptomic response of D. magna exposed to sublethal doses of 1H-benzotriazole (BTR), 5-methyl-1H-benzotriazole (5MeBTR) and 5-chloro-1H-benzotriazole (5ClBTR) using RNA-sequencing and quantitative real-time PCR. Cellular and life-history endpoints (survival, number of neonates, growth) were also investigated. Significant effects on the molting frequency were observed after 21-d exposure to 5MeBTR and 5ClBTR. No effects on molting frequency were observed for BTR but RNA-seq results indicated that this BZT induced the up-regulation of genes coding for cuticular proteins, which could have compensated the molting disruption. Molting in cladocerans is actively controlled by ecdysteroid hormones. Complementary short-term temporal analysis (4- and 8-d exposure) of the transcription of genes related to molting and hormone-mediated processes indicated that the three compounds had specific modes of action. BTR induced the transcription of genes involved in 20-hydroxyecdysone synthesis, which suggests pro-ecdysteroid properties. 5ClBTR exposure induced protein activity and transcriptional levels of chitinase enzymes, associated with an impact on ecdysteroid signaling pathways, which could explain the decrease in molt frequency. Finally, 5MeBTR seemed to increase molt frequency through epigenetic processes. Overall, results suggested that molting effects observed at the physiological level could be linked to endocrine regulation impacts of BZTs at the molecular level.

Endocrine-disruption potential of perfluoroethylcyclohexane sulfonate (PFECHS) in chronically exposed Daphnia magna.

Perfluoroethylcyclohexane sulfonate (PFECHS), mainly used in hydraulic fluids in aircrafts, is a member of the perfluoroalkyl sulfonate family which includes the regulated perfluorooctane sulfonate (PFOS). PFECHS has been reported in environmental samples but its toxicity to aquatic organisms is unknown. The objectives of this study were to identify biological pathways altered by sublethal exposure (12 d) of D. magna to PFECHS (0.06, 0.6, and 6 mg/L) using microarray and quantitative real-time PCR and to identify potential biomarkers to link transcriptomic to phenotypic responses. PFECHS was also quantified in surface water samples (1.04-1.38 ng/L) collected from the St. Lawrence River, Canada. Transcriptomic analyses indicated the under-regulation of vitellogenin-related genes (VTG1) in PFECHS-exposed groups. PFECHS exposure also led to the up-regulation of genes related to cuticle. VTG was selected as a potential cellular marker and identified in D. magna using an immuno-specific assay and quantified using Western blot and LC/MS/MS. Results indicated a decrease of VTG content in exposed D. magna which was in concordance with the transcription of VTG-related genes. No effects were observed on survival, molting, or reproduction at the individual/population levels. Overall, results suggest endocrine disruption potential for PFECHS in D. magna at concentrations higher than levels reported in the aquatic environment.

Integrated spatial health assessment of yellow perch (Perca flavescens) populations from the St. Lawrence River (QC, Canada), part B: cellular and transcriptomic effects.

Multi-biological level assessments have become great tools to evaluate the health of aquatic ecosystems. Using this approach, a complementary study was designed to evaluate the health of yellow perch (Perca flavescens) populations in the St. Lawrence River (Quebec, Canada). In the present study, stress responses were compared at the transcriptomic, cellular, and tissue levels in yellow perch collected at six sites along the river: Lake St. François, Lake St. Louis (north and south), Beauregard Island and Lake St. Pierre (north and south). These results complement the physiological and chemical parameters as well as pathogen infection investigated in a companion paper published in the present issue. Thiobarbituric acid reactive substance (TBARS) analyses indicated the presence of oxidative stress in fish collected in the southern part of Lake St. Louis and at the downstream sites of Lake St. Pierre. High lipid peroxidation levels were found in the muscle of yellow perch caught at Beauregard Island, located downstream of the Montreal's wastewater treatment plant, suggesting an impact of the municipal effluent on redox homeostasis. Transcriptomic results indicated the down-regulation of genes related to lipid, glucose, and retinoid in southern Lake St. Pierre as well as a decrease in retinoid storage. Overall, biochemical and molecular markers indicated that the health status of yellow perch followed a decreasing gradient from upstream to downstream of the St. Lawrence River. This gradient is representative of the cumulative negative impacts of human activities on water and habitat quality along the river.

Chronic toxicity evaluation of the flame retardant tris (2-butoxyethyl) phosphate (TBOEP) using Daphnia magna transcriptomic response.

Tris (2-butoxyethyl) phosphate (TBOEP) is an organophosphorous-containing flame retardant (OPFR) of high production volume used in a broad range of applications. The use of TBOEP containing products has resulted in its release and ubiquitous occurrence in the aquatic environment. In this study, Daphnia magna transcriptomic response was measured by microarray to evaluate sublethal effects of TBOEP as part of a multi-level biological approach including specific gene transcription measured by qRT-PCR, enzyme activity, and life-history endpoints (i.e., survival, growth and reproduction). Chronic exposure (21 d) to a range of sublethal concentrations of TBOEP (14.7-1470µgL(-1)) did not impact growth, survival or reproduction, although the number of offspring decreased between the lowest and the highest dose. Gene transcription profiling by microarray analysis revealed that 101 genes were differentially transcribed in response to TBOEP (fold change treated/control ±1, p<0.05). Most of the responding genes were involved in protein metabolism (9), biosynthesis (4) and energy metabolism (6) indicating that TBOEP could have chronic toxic effects on aquatic organisms at sublethal doses by disrupting essential biological pathways. Nine genes were found to be commonly affected by more than one dose, including a gene coding for cathepsin D and multiple isoforms of genes coding for hemoglobin, suggesting potential biomarkers of interest. Microarray results were confirmed by qRT-PCR and measurements at the protein level as cathepsin D enzymatic activity increased significantly in the highest dose treatment. Results highlight the relevance of using the transcriptomic response of D. magna as a first line of evidence to unravel the mode of action of chemicals.

Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters.

Trichloroethylene (TCE) is a ubiquitous contaminant classified as a human carcinogen. Vinyl chloride (VC) is primarily used to manufacture polyvinyl chloride and can also be a degradation product of TCE. Very few data exist on the toxicity of TCE and VC in aquatic organisms particularly at environmentally relevant concentrations. The aim of this study was to evaluate the sub-lethal effects (10 day exposure; 0.1; 1; 10 µg/L) of TCE and VC in Daphnia magna at the gene, cellular, and life-history levels. Results indicated impacts of VC on the regulation of genes related to glutathione-S-transferase (GST), juvenile hormone esterase (JHE), and the vitelline outer layer membrane protein (VMO1). On the cellular level, exposure to 0.1, 1, and 10 µg/L of VC significantly increased the activity of JHE in D. magna and TCE increased the activity of chitinase (at 1 and 10 µg/L). Results for life-history parameters indicated a possible tendency of TCE to affect the number of molts at the individual level in D. magna (p=0.051). Measurement of VG-like proteins using the alkali-labile phosphates (ALP) assay did not show differences between TCE treated organisms and controls. However, semi-quantitative measurement using gradient gel electrophoresis (213-218 kDa) indicated significant decrease in VG-like protein levels following exposure to TCE at all three concentrations. Overall, results indicate effects of TCE and VC on genes and proteins related to metabolism, reproduction, and growth in D. magna.

Transcriptional and cellular responses of the green alga Chlamydomonas reinhardtii to perfluoroalkyl phosphonic acids.

Perfluoroalkyl phosphonic acids (PFPAs), a new class of perfluoroalkyl substances used primarily in the industrial sector as surfactants, were recently detected in surface water and wastewater treatment plant effluents. Toxicological effects of PFPAs have as yet not been investigated in aquatic organisms. The objective of the present study was to evaluate the effects of perfluorooctylphosphonic acid (C8-PFPA) and perfluorodecylphosphonic acid (C10-PFPA) exposure (31-250µg/L) on Chlamydomonas reinhardtii using genomic (qRT-PCR), biochemical (reactive oxygen species production (ROS) and lipid peroxidation), and physiological (cellular viability) indicators. After 72h of exposure, no differences were observed in cellular viability for any of the two perfluorochemicals. However, increase in ROS concentrations (36% and 25.6% at 125 and 250µg/L, respectively) and lipid peroxidation (35.5% and 35.7% at 125 and 250µg/L, respectively) was observed following exposure to C10-PFPA. C8-PFPA exposure did not impact ROS production and lipid peroxidation in algae. To get insights into the molecular response and modes of action of PFPA toxicity, qRT-PCR-based assays were performed to analyze the transcription of genes related to antioxidant responses including superoxide dismutase (SOD-1), glutathione peroxidase (GPX), catalase (CAT), glutathione S-transferase (GST), and ascorbate peroxidase (APX I). Genomic analyses revealed that the transcription of CAT and APX I was up-regulated for all the C10-PFPA concentrations. In addition, PFPAs were quantified in St. Lawrence River surface water samples and detected at concentrations ranging from 250 to 850pg/L for C8-PFPA and 380 to 650pg/L for C10-PFPA. This study supports the prevalence of PFPAs in the aquatic environment and suggests potential impacts of PFPA exposure on the antioxidant defensive system in C. reinhardtii.

A multi-level biological approach to evaluate impacts of a major municipal effluent in wild St. Lawrence River yellow perch (Perca flavescens).

The development of integrated ecotoxicological approaches is of great interest in the investigation of global concerns such as impacts of municipal wastewater effluents on aquatic ecosystems. The objective of this study was to investigate the effects of a major wastewater municipal effluent on fish using a multi-level biological approach, from gene transcription and enzyme activities to histological changes. Yellow perch (Perca flavescens) were selected based on their wide distribution, their commercial and recreational importance, and the availability of a customized microarray. Yellow perch were sampled upstream of a major municipal wastewater treatment plant (WWTP) and 4 km and 10 km downstream from its point of discharge in the St. Lawrence River (Quebec, Canada). Concentrations of perfluoroalkyl substances (PFASs), polybrominated diphenyl ethers (PBDEs) and metals/trace elements in whole body homogenates were comparable to those from other industrialized regions of the world. Genomic results indicated that the transcription level of 177 genes was significantly different (p<0.024) between exposed and non-exposed fish. Among these genes, 38 were found to be differentially transcribed at both downstream sites. Impacted genes were associated with biological processes and molecular functions such as immunity, detoxification, lipid metabolism/energy homeostasis (e.g., peroxisome proliferation), and retinol metabolism suggesting impact of WWTP on these systems. Moreover, antioxidant enzyme activities were more elevated in perch collected at the 4 km site. Biomarkers of lipid metabolism, biosynthetic activity, and aerobic capacities were significantly lower (p<0.05) in fish residing near the outfall of the effluent. Histological examination of the liver indicated no differences between sites. Correlations between PFAS, PBDE, and metal/trace element tissue concentrations and markers of peroxisomal proliferation, oxidative stress, and retinoid metabolism were found at the gene and cellular levels. Present results suggest that relating transcriptomic analyses to phenotypic responses is important to better understand impacts of environmental contamination on wild fish populations.

Sublethal effects of the flame retardant intermediate hexachlorocyclopentadiene (HCCPD) on the gene transcription and protein activity of Daphnia magna.

Hexachlorocyclopentadiene (HCCPD) is a chlorinated chemical of high production volume used as an intermediate in the production of flame retardants. HCCPD may be released to the environment during production, use, and as a result of product degradation. The objectives of this study were to evaluate sublethal effects of HCCPD exposure to Daphnia magna at environmentally relevant concentrations (0.0138-13.8 µg/L) using genomic tools (microarray and qPCR), enzyme activities, and life-history endpoints (survival, reproduction, and growth). In chronic exposures, no differences were observed in life-history endpoints (survival, time of first brood, time of first molt, molt frequency, number of neonates, and body length) between exposed organisms and controls. Microarray analyses indicated significant differential genomic transcription for 46 genes (p-value ≤ 0.05 and fold-change>2). Five identified genes were related to metabolic functions. Enzyme activities of α-amylase and trypsin, selected based on transcriptional responses, were evaluated in D. magna. Although trypsin activity was similar between treatments and controls, the activity of α-amylase significantly decreased with increasing HCCPD concentrations. On the chemical level, instability of HCCPD was observed in spiked culture media, most probably due to photolysis and biodegradation. HCCPD was not detected in surface water samples collected upstream and at the point of discharge of a major wastewater treatment plant effluent. Environmentally, rapid degradation of HCCPD could be outdone by its continuous release into aquatic ecosystems in specific areas of concern (e.g., vicinity of industries and hazardous sites). Toxicity results from this study highlight the use of genomics in the identification of biomarkers and help advance the science, and potential use, of multi-level biological approaches for environmental risk assessment.

Induction of gene responses in St. Lawrence River northern pike (Esox lucius) environmentally exposed to perfluorinated compounds.

Municipal waste water effluents (MWWEs) are important sources of chemical contamination for aquatic environments. This study investigated the presence and effects of perfluorinated compounds (PFCs) in environmentally exposed northern pike (Esox lucius) collected upstream and downstream a major municipal waste water treatment plant (WWTP) in the St. Lawrence River, Canada. Twelve PFCs, including the newly detected perfluoroethylcyclohexane sulfonate (PFECHS), were quantified in fish muscle, liver, and plasma. Additionally, the expression of eight genes and the activity of three biomarkers were analyzed in fish tissues at both sites. Mean ∑PFC concentration in fish plasma collected upstream the WWTP was 185ng/g w.w. compared to 545ng/g w.w. downstream the point of release. PFECHS was quantified for the first time in St. Lawrence River fish (mean plasma concentration in MWWE fish: 5.07±4.72ng/g w.w.). Results of transcriptomic responses were tissue-specific and indicated significant up-regulation for metallothionein (MT) in blood and MT, glutathion-S-transferase (GST), superoxide dismutase (SOD), and cytochromes P450 1A1 (CYP1A1) in gill tissue of fish collected in the MWWE suggesting greater stress responses for organisms at this location. Significant relationships were found between vitellogenin (Vtg) gene expression in liver, Vtg activity in plasma and perfluorotridecanoic acid (PFTrA), perfluorotetradecanoic acid (PFTeA), and perfluorodecane sulfonate (PFDS) plasma concentrations. The possible endocrine effects of these PFCs should be further investigated.