Transcriptome analyses in juvenile yellow perch (Perca flavescens) exposed in vivo to clothianidin and chlorantraniliprole: Possible sampling bias.

The St. Lawrence River is an important North American waterway that is subject to anthropogenic pressures including intensive urbanization, and agricultural development. Pesticides are widely used for agricultural activities in fields surrounding the yellow perch (Perca flavescens) habitat in Lake St. Pierre (Quebec, Canada), a fluvial lake of the river where the perch population has collapsed. Clothianidin and chlorantraniliprole were two of the most detected insecticides in surface waters near perch spawning areas. The objectives of the present study were to evaluate the transcriptional and biochemical effects of these two pesticides on juvenile yellow perch exposed for 28d to environmental doses of each compound alone and in a mixture under laboratory/aquaria conditions. Hepatic mRNA-sequencing revealed an effect of chlorantraniliprole alone (37 genes) and combined with clothianidin (251 genes), but no effects of clothianidin alone were observed in perch. Dysregulated genes were mostly related to circadian rhythms and to Ca2+ signaling, the latter effect has been previously associated with chlorantraniliprole mode of action in insects. Moreover, chronic exposure to clothianidin increased the activity of acetylcholinesterase in the brain of exposed fish, suggesting a potential non-target effect of this insecticide. Further analyses of three clock genes by qRT-PCR suggested that part of the observed effects of chlorantraniliprole on the circadian gene regulation of juvenile perch could be the result of time-of-day of sacrifice. These results provide insight into biological effects of insecticides in juvenile perch and highlight the importance of considering the circadian rhythm in experimental design and results analyses.

Associations between total mercury, trace minerals, and blood health markers in Northwest Atlantic white sharks (Carcharodon carcharias).

The ecology and life-histories of white sharks make this species susceptible to mercury bioaccumulation; however, the health consequences of mercury exposure are understudied. We measured muscle and plasma total mercury (THg), health markers, and trace minerals in Northwest Atlantic white sharks. THg in muscle tissue averaged 10.0 mg/kg dry weight, while THg in blood plasma averaged 533 µg/L. THg levels in plasma and muscle were positively correlated with shark precaudal length (153-419 cm), and THg was bioaccumulated proportionally in muscle and plasma. Nine sharks had selenium:mercury molar ratios in blood plasma >1.0, indicating that for certain individuals the potential protective effects of the trace mineral were diminished, whereas excess selenium may have protected other individuals. No relationships between plasma THg and any trace minerals or health markers were identified. Thus, we found no evidence of negative effects of Hg bioaccumulation, even in sharks with very high THg.

On the Fate of Butyl Methoxydibenzoylmethane (Avobenzone) in Coral Tissue and Its Effect on Coral Metabolome.

The intensive use of sunscreen products has raised concerns regarding their environmental toxicity and the adverse impacts of ultraviolet (UV) filters on ecologically important coral communities. Prior metabolomic analyses on symbiotic coral Pocillopora damicornis exposed to the UV filter butyl methoxydibenzoylmethane (BM, avobenzone) revealed unidentified ions in the holobiont metabolome. In the present study, follow-up differential metabolomic analyses in BM-exposed P. damicornis detected 57 ions with significantly different relative concentrations in exposed corals. The results showed an accumulation of 17 BM derivatives produced through BM reduction and esterification. The major derivative identified C16:0-dihydroBM, which was synthesized and used as a standard to quantify BM derivatives in coral extracts. The results indicated that relative amounts of BM derivatives made up to 95% of the total BM (w/w) absorbed in coral tissue after 7 days of exposure. Among the remaining metabolites annotated, seven compounds significantly affected by BM exposure could be attributed to the coral dinoflagellate symbiont, indicating that BM exposure might impair the photosynthetic capacity of the holobiont. The present results suggest that the potential role of BM in coral bleaching in anthropogenic areas should be investigated and that BM derivatives should be considered in future assessments on the fate and effects of BM in the environment.

Flame retardants and their associations with thyroid hormone-related variables in northern fulmars from the Faroe Islands.

Flame retardants (FRs) are widely reported in tissues of seabirds including birds sampled from remote areas. There is evidence that FRs can disrupt the hypothalamic-pituitary-thyroid (HPT) axis in seabirds, although information is limited on thyroid-related mechanisms and effects. This study investigated the associations between concentrations of polybrominated diphenyl ethers (PBDEs) and other FRs, and changes in the HPT axis in northern fulmars (Fulmarus glacialis) from the Faroe Islands (North Atlantic). Plasma concentrations of thyroid hormones (THs), hepatic deiodinase type 1 (D1) activity, and transcription of selected TH-related genes in liver were used as markers of HPT axis changes. Liver concentrations of a certain PBDE congeners and other FRs including pentabromoethylbenzene (PBEB), dechlorane 602 (Dec-602), and dechlorane plus (DP) were associated with changes in thyroid status. Specifically, liver PBDE, PBEB and Dec-602 concentrations were associated with plasma TH levels (free thyroxine [FT4] and total triiodothyronine [TT3]). Liver DP concentrations were positively correlated with the TT4:FT4 ratios and mRNA levels of UDP-glucuronyltransferase-1, while those of PBEB were negatively associated with TT4:TT3 ratios and D1 activity. D1 activity was also positively associated with the tri-, tetra- and hexa-BDE congeners. Moreover, transcription of ABCC2, a hepatic TH transporter, was associated with certain liver PBDE concentrations. Although PBDEs and other FRs may be potential inhibitors of D1 activity, only a few of the targeted FRs had modest associations with hepatic D1 activity. Regardless, the relationships reported herein indicated that exposure to moderate levels of FRs can be associated with thyroid axis perturbation at the molecular/biochemical levels in this North Atlantic seabird species.

An investigation of physiological effects of the Deepwater Horizon oil spill on a long-distance migratory seabird, the northern gannet.

Exposure to oil can have long-term impacts on migratory birds. Following the 2010 Deepwater Horizon blowout in the Gulf of Mexico (GOM), we investigated potential impacts of oil exposure on a population of northern gannets (Morus bassanus) that breed on Bonaventure Island (Québec, Canada) and winter in GOM and along the U.S. Atlantic coast (AC). Blood and feather samples were collected from adults previously equipped with geolocators to determine wintering locations. Parent and alkylated polycyclic aromatic hydrocarbons (PAHs); trace metals; stable isotopes of carbon, nitrogen, and hydrogen; and immune, thyroid, steroid, retinoid, and genetic endpoints were measured. PAH and trace metal concentrations did not differ between gannets using different wintering sites. Feather stable isotope values varied significantly between birds from different wintering locations. Gannets wintering in GOM showed higher feather corticosterone and plasma thyroid hormone levels, which may indicate increased energetic demands and/or greater exposure to environmental stressors.

Food-Borne Exposure of Juvenile Rainbow Trout (Oncorhynchus mykiss) to Benzotriazole Ultraviolet Stabilizers Alone and in Mixture Induces Specific Transcriptional Changes.

Benzotriazole ultraviolet-stabilizers (BZT-UVs) are commonly used as additives to protect from light-induced degradation in a variety of consumer goods. Despite their widespread presence in aquatic ecosystems, information on the effects of these compounds remains largely unknown. The objectives of the present study were to evaluate the chronic effects of 2 BZT-UVs alone and in a mixture, 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol (UV-234) and 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV-328), in juvenile rainbow trout (Oncorhynchus mykiss) chronically exposed (for 28 d) through the diet. Chemical analyses of livers from exposed trout suggested liver accumulation and potential metabolism of the 2 compounds. Hepatic RNA-sequencing analyses revealed specific effects of each compound on gene transcription profiles; UV-234 affected mainly genes involved in cellular metabolism, whereas UV-328 induced the transcription of ribosomal proteins and downregulated genes involved in immune responses. Both compounds regulated iron homeostasis genes in an opposite manner. The mixture of both BZT-UVs did not produce significant evidence of additive or synergistic effects. Environ Toxicol Chem 2020;39:852-862. © 2020 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry © 2020 SETAC.

Cumulative effects of municipal effluent and parasite infection in yellow perch: A field study using high-throughput RNA-sequencing.

Multiple metabolic, immune and reproductive effects have been reported in fish residing in effluent-impacted sites. Natural stressors such as parasites also have been shown to impact the responses of organisms to chronic exposure to municipal effluent in the St. Lawrence River (Quebec, Canada). In order to comprehensively evaluate the cumulative impacts of anthropogenic and natural stressors on the health of yellow perch, differential mRNA transcription profiles were examined in juvenile females collected from effluent-impacted and upstream sites with low or high infection levels of the larval trematode Apophallus brevis. Transcriptomics was used to identify biological pathways associated with environmental exposure. In total, 3463 isoforms were differentially transcribed between sites. Patterns reflecting the combined effects of stressors were numerically dominant, with a majority of downregulated transcripts (68%). The differentially expressed transcripts were associated with 27 molecular and cellular functions ranging from cellular development to xenobiotic metabolism and were involved in the development and function of 13 organ systems including hematological, hepatic, nervous, reproductive and endocrine systems. Based on RNA-seq results, sixteen genes were measured by qPCR. Significant differences were observed for six genes in fish exposed to both stressors combined, whereas parasites and effluent individually impacted the transcription of one gene. Lysozyme activity, lipid peroxidation, retinol-binding protein and glucose-6-phosphate dehydrogenase were selected as potential biomarkers of effects to study specific pathways of interest. Lipid peroxidation in perch liver was different between sites, parasite loads, and for combined stressors. Overall, results indicated that juvenile yellow perch responded strongly to combined parasite and effluent exposure, suggesting cumulative effects on immune responses, inflammation and lipid metabolism mediated by retinoid receptors. The present study highlight the importance of using a comprehensive approach combining transcriptomics and endpoints measured at higher levels of biological organization to better understand cumulative risks of contaminants and pathogens in aquatic ecosystems.

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.

Transcriptional and cellular effects of benzotriazole UV stabilizers UV-234 and UV-328 in the freshwater invertebrates Chlamydomonas reinhardtii and Daphnia magna.

Benzotriazole ultra violet stabilizers (BZT-UVs) are compounds used in many applications and products to prevent photochemical degradation. Despite their widespread presence in aquatic ecosystems and persistence in the environment, there are very limited data on their effects and toxicity, and their modes of action remain largely unknown. The objectives of the present study were to evaluate the chronic effects of 2 BZT-UVs, 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol (UV-234) and 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV-328), on the freshwater green algae Chlamydomonas reinhardtii and the freshwater crustacean Daphnia magna. Organisms were exposed to 0.01 and 10 µg/L of UV-234, UV-328, as well as a mixture of the 2 compounds. Life-history endpoints (viability, reproduction, and growth) and oxidative stress-related biomarkers (gene transcription, reactive oxygen species [ROS] production, and lipid peroxidation) were measured. Daphnia magna growth, reproduction, and gene transcription were not impacted by 21-d individual or mixed exposure. After 96-h of exposure, no differences were observed on the cellular viability of C. reinhardtii for either of the 2 BZT-UVs. In the algae, results showed increased ROS production in response to UV-328 and lipid peroxidation following exposure to UV-234. Synergistic effects of the 2 BZT-UVs were evident at the transcriptional level with 2 to 6 times up-regulation of glutathione peroxidase (gpx ) in response to the mixture for all treatment conditions. The transcription of superoxide dismutase (sod), catalase (cat), and ascorbic peroxidase (apx) was also regulated by UV-234 and UV-328 in the green algae, most likely as a result of ROS production and lipid peroxidation. Results from the present study suggest potential impacts of UV-234 and UV-328 exposure on the antioxidant defense system in C. reinhardtii. Environ Toxicol Chem 2017;36:3333-3342. © 2017 Crown in the Right of Canada. Published by Wiley Periodicals Inc., on behalf of SETAC.

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.

Integrated spatial health assessment of yellow perch (Perca flavescens) populations from the St. Lawrence River, Quebec, Canada) part A: physiological parameters and pathogen assessment.

A multi-disciplinary approach was used to evaluate the health of yellow perch (Perca flavescens) in the St. Lawrence River (Quebec, Canada), which is experiencing a severe population decline in the downstream portion of the river. Physiological parameters, liver alterations, trace metal concentrations, parasite prevalence and abundance, stable isotope composition, and the presence/absence of the viral hemorragic septicemia virus (VHSV) were evaluated in 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). Trace metal concentrations in surface water were higher in Lake St. Louis and downstream of a major urban wastewater treatment plant discharge, indicating that this effluent was a significant source of Cu, As, Ag, Zn, and Cd. Levels of Pb in surface water exceeded thresholds for the protection of aquatic life in Lake St. Louis and were negatively correlated with body condition index in this lake. In Lake St. Pierre, Cu, Ag, and Cd bioaccumulated significantly in perch liver and lower body condition index and greater liver damage were observed compared to upstream sites. Parasite analyses indicated a higher abundance of metacercariae of the trematodes Apophallus brevis and Diplostomum spp. in Lake St. Louis, and VHSV was not detected in the liver of yellow perch for all studied sites. Overall, results suggested that the global health of yellow perch from Lake St. Pierre is lower compared to upstream studied sites, which could contribute to the documented population collapse at this site.

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.

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.

Nuclear receptors HR96 and ultraspiracle from the fall armyworm (Spodoptera frugiperda), developmental expression and induction by xenobiotics.

The fall armyworm Spodoptera frugiperda is a major polyphagous pest in agriculture and little is known on how this insect can adapt to the diverse and potentially toxic plant allelochemicals that they ingest or to insecticides. To investigate the involvement of nuclear receptors in the response of S. frugiperda to its chemical environment, we cloned SfHR96, a nuclear receptor orthologous to the mammalian xenobiotic receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). We also cloned ultraspiracle (USP), the ortholog of retinoid X receptor (RXR) that serves as partner of dimerization of PXR and CAR. Cloning of SfUSP revealed the presence of two isoforms, SfUSP-1 and SfUSP-2 in this species, that differ in their N-terminal region. The expression of these receptors as well as the ecdysone receptor was studied during specific steps of development in different tissues. SfHR96 was constitutively expressed in larval midgut, fat body and Malpighian tubules throughout the last two instars and pupal stage, as well as in Sf9 cells. EcR and SfUSP-2 showed peaks of expression before larval moults and during metamorphosis, whereas SfUSP-1 was mainly expressed in the pre-pupal stage. Receptor induction was followed after exposure of larvae or cells to 11 chemical compounds. SfHR96 was not inducible by the tested compounds. EcR was significantly induced by the 20-hydroxyecdysone agonist, methoxyfenozide, and SfUSP showed an increase expression when exposed to the juvenile hormone analog, methoprene. The cloning of these nuclear receptors is a first step in understanding the important capacities of adaptation of this insect pest.

Effects of hormone agonists on Sf9 cells, proliferation and cell cycle arrest.

Methoxyfenozide and methoprene are two insecticides that mimic the action of the main hormones involved in the control of insect growth and development, 20-hydroxyecdysone and juvenile hormone. We investigated their effect on the Spodoptera frugiperda Sf9 cell line. Methoxyfenozide was more toxic than methoprene in cell viability tests and more potent in the inhibition of cellular proliferation. Cell growth arrest occurred in the G2/M phase after a methoprene treatment and more modestly in G1 after methoxyfenozide treatment. Microarray experiments and real-time quantitative PCR to follow the expression of nuclear receptors ultraspiracle and ecdysone receptor were performed to understand the molecular action of these hormone agonists. Twenty-six genes were differentially expressed after methoxyfenozide treatment and 55 genes after methoprene treatment with no gene in common between the two treatments. Our results suggest two different signalling pathways in Sf9 cells.

Regulation of cytochrome P450 expression in Drosophila: Genomic insights.

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the "phenobarbital-type" induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from "detox" microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.