ABSTRACT: To determine the effects on the intestine gene expression of pathogen exposure to Enteromyxum leei. One fish group was exposed to E. leei-contaminated effluent (recipient group = R) . R fish (n= 66, average weight = 134 g) were placed in two replicated 200L fibre-glass tanks which were set to receive exclusively the effluent water from another tank containing 24 infected (donors = D; average weight = 127.3 g; prevalence of infection = 54%) gilthead sea bream. The D to R fish ratio was 0.8. Other 66 naïve fish were allocated in two replicated tanks (control group = CTRL) under the same conditions, but without receiving contaminated effluent. Over the course of the study, day length followed natural changes and water was heated in order to keep temperature always above 18ºC, the range was 18-23 ºC. Water was 5 µm-filtered and UV irradiated, and salinity was 37.5‰. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight. Disease signs and daily mortalities were recorded throughout the experiments. The parasitic status of dead fish was checked by microscopic examination of fresh intestinal scrapings. Fish were sampled after 113 days post exposure (p.e.). Feeding was stopped one day prior to the sampling to ensure that the digestive tract was empty. Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80 °C until RNA extraction and analysis. Keywords: Treated/Untreated, confinement, cortisol, stress response, time course, microarray 28 intestine samples - Twenty eight slides were hybridised using a reference design. Three groups (control, infected and non-infected) were compared using five individual fish in each group. Each sample was hybridised twice - the second being a dye-swap of the first.
SUBMITTER: Anita Talbot Grace Davey Michael Taylor Cairns Jaume Pérez-Sánchez Josep Calduch-Giner Michael T Cairns Richard Reinhardt Benoit Houeix
Project description:To determine the effects of pathogen exposure to Enteromyxum leei on head kidney gene expression. Keywords: confinement, cortisol, stress response, time course, microarray 30 head kidney samples - Thirty slides were hybridised using a reference design. Three groups (control, infected and non-infected) were compared using five individual fish in each group. Each sample was hybridised twice - the second being a dye-swap of the first.
Project description:Rivers containing effluents from water treatment plants are complex soups of compounds, ranging from pharmaceuticals to natural hormones. Male fathead minnows (Pimephales promelas) were exposed for 3 weeks to effluent waters from the Metropolitan Wastewater Treatment Plant in St. Paul, MN. Fish were tested for their competitive nest holding behavior. Changes in vitellogenin were measured and these were correlated to changes in gene expression using a 22,000 gene microarray developed specifically for fathead minnows. Significant changes in gene expression were observed in both liver and gonad, which correlate to phenotypic changes of vitellogenin induction and reduced competitive behavior. We also compared by real-time PCR the expression changes in key genes related to steroid biosynthesis and metabolism in fish exposed to the effluent as well as in fish exposed to a model estrogen and a model androgen. While the gene expression signature from effluent-exposed fish shared some elements with estrogen and androgen signatures, overall it was different, underscoring the complexity of compounds present in sewage and their different modes of action. Fathead minnow 22,000 gene arrays were designed by EcoArray (Alachua, FL) and were purchased from Agilent. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference consisted of equal amounts of RNA from control female and male tissues (liver, brain and gonad) and was prepared as a standard for several experiments. Four replicates consisting of four different individuals were analyzed for each of the tissues, liver and gonad for both the control and the effluent-exposed fish. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturer’s kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA). Ovarian and liver samples from the fish were labeled with Cy5 while the reference sample was labeled with Cy3.
Project description:Despite recent knowledge of the potential environmental impact that compounds present in municipal wastewater effluents, including contaminants of emerging concern (CECs), may have, the implications of fish exposure to this contaminant mixtures are not completely understood. The effects caused by effluent CECs may be subtle and diverse, thus the need for sensitive and comprehensive tools such as gene expression to detect such responses. In this study, we conducted laboratory exposures that examined plasma concentrations of vitellogenin (VTG), changes in secondary sexual characteristics and gene expression in sexually mature male fathead minnows (Pimephales promelas) exposed to environmentally realistic (0.5%) and higher (5%) concentrations of municipal wastewater effluents. Secondary and primary treated effluents were used. Several of the 32 CECs investigated were detected, including pharmaceuticals, personal care products, hormones, current use pesticides and industrial compounds. The percent of males with detectable levels of VTG was higher in fish exposed to effluent treatments. An increased number of males with changes in secondary sexual characteristics (e.g. development of ovipositors), was observed in fish exposed to 5% effluent treatments. Gene expression data indicated that overall expression patterns were characteristic to each effluent. Higher numbers of differentially expressed genes were observed in fish exposed to primary treated effluent when compared to controls. Differentially expressed genes belonged to several functional categories, including xenobiotic metabolism, estogenicity and energy/metabolism processes. Gene expression data provided information to understand some of the mechanisms behind the effects observed at higher biological levels. To investigate gene expression responses resulting from exposure to POTW effluents, two laboratory experiments were conducted using effluent from San Diego (Point Loma; SD) and Los Angeles (Hyperion; LA). The LA effluent received secondary treatment and the SD effluent received advanced primary treatment. Treatments used during exposures consisted of negative controls (moderately hard water), positive controls (E2), and 0.5% and 5% effluent concentrations. The 0.5% concentration of effluent represented an environmentally realistic exposure level. The 5% effluent concentration represented a higher level at which we expected biological responses. The exposures lasted 14 days. Treatments: EFFHa = 5% primary treated effluent EFFHb = 5% secondary treated effluent EFFLa = 0.5% primary treated effluent E2a = Estradiol, positive control for primary effluent E2b = Estradiol, positive control for secondary effluent CTRLa = Moderately hard water, negative control for primary effluent CTRLb = Moderately hard water, negative control for secondary effluent
Project description:To assess the impact of a municiple effluent across different environments, a gradient design (upstream, downstream, and at effluent) was set up across three waste water treatment plant outflows in three different regions of MN. These three sites represents vastly different land use and contamination profiles. The upstream location at each site was used as a point of comparison to reduce site specific differences in water. Fish were exposed at three sites in three locations for 4 days. The objectives of the study were to 1). determine if biological impact of the effluent could be detected at the downstream site; 2). If the use of the upstream site would allow point source influence to be detected over the ambient level of biological activity; 3). if using functional analyses of transcriptomic results would show similarities between effleunt and downstream sites. The current series contains n=53 microarrays associated with fish exposed at three locations (upstream, effluent, downstream) at 3 different site R, H, E for 4 days until tissues were collected At the specific locations (UP, EF, DS), sexually mature female fathead minnows (SI-SM2) were exposed to location specific water in mini-mobile environmental monitoring units (MMU)14 for a period of 4d, at each of three sites (R, H, E) during the summer and fall of 2010 (SI-SM3). The MMUs allowed for consistency in aeration, temperature, and feeding. In the case of the riverine R and H sites, the MMU was supplied with a continuous flow of location-specific water. Due to logistical constraints, exposures at the lake site (E) were conducted under static renewal conditions within the MMUs. However, the cumulative number of daily water exchanges were equivalent to those in the flow-through studies. After exposure fish were anesthetized (buffered MS-222), and necropsied at a facility near the site, specific tissues were excised and flash frozen in liquid nitrogen, and stored at -80°C. Gonads were removed from female and flash-frozen for gene expression analyses using microarray. Ovarian transcripts from six females per location (three locations per site, three sites) were analyzed using a custom 15,000 feature microarray (GEO Platform Accession GPL9248). Data sets for this phase (n=53 microarrays) were normalized independently using Fastlo (Ballman et al., 2004) implemented in R (http://www.r-project.org/), but analyzed using parallel approaches.
Project description:Municipal wastewater effluent can impact its receiving environment. In the St. Lawrence River, male fish living downstream from Montreal exhibit increased hepatic vitellogenin, intersex, delayed spermatogenesis and altered immune function. Few studies have examined genome-wide effects associated with municipal effluent exposure in fish to decipher the mechanisms of toxicity. The present objective was to identify hepatic cellular signaling pathways in fathead minnows following exposure to municipal wastewater effluent. Immature minnows were exposed for 21 days to either 0% (Control) or 20% municipal effluent, the highest concentration in the St. Lawrence River. Hepatic RNA was extracted and used to hybridize a fathead minnow oligonucleotide microarray containing approximately 15K gene sequences. Sixteen samples were examined, 8 control samples and 8 exposed samples.
Project description:Patancheru, near Hyderabad, India, is a major production site for the global bulk drug market. About 90 manufacturers send their wastewater to a common treatment plant in Patancheru. Extraordinary high levels of a wide range of pharmaceuticals have recently been demonstrated in the treated effluent. As little as 0.2% of this effluent can strongly reduce the growth rate of tadpoles, but the underlying mechanisms of toxicity are not known. To begin addressing how the effluent affects aquatic vertebrates, rainbow trout (Oncorhynchus mykiss) were exposed to 0.2% effluent for five days. Several physiological endpoints, together with effects on global hepatic gene expression patterns, were analyzed. The exposed fish showed both an induction of hepatic cytochrome P450 1A (CYP1A) gene expression, as well as EROD activity. Clinical blood chemistry analyses revealed an increase in plasma phosphate levels, which in humans indicates impaired kidney function. Several oxidative stress-related genes were induced in the livers, however, no significant changes in antioxidant enzyme activities or in the hepatic glutathione levels were found. Furthermore, estrogen-regulated genes were slightly up-regulated following exposure, and moderate levels of estriol were detected in the effluent. This study identifies changes in gene expression triggered by exposure to a high dilution of the effluent, supporting the hypothesis that these fish are responding to chemical exposure. The pattern of regulated genes may contribute to the identification of mechanisms of sub-lethal toxicity, as well as illuminate possible causative agents. Two exposure experiments were conducted with control fish and fish exposed to 0.2% effluent. 19 pools of RNA, each containing three fish, were hybrdizied to in situ synthesized custom-made oligonucleotide microarrays. In total, four exposed and four control pools from exposure experiment I and five exposed and six control pools from exposure experiment II were analyzed.
Project description:Laboratory tests with marine flatfish were conducted to investigate associations among gene expression, higher biological responses and wastewater effluent exposure. Previous studies showed molecular responses such as elevated concentrations of plasma estradiol and vitellogenin in wild male hornyhead turbot (Pleuronichthys verticalis). In the present study, male hornyhead turbot were exposed to environmentally realistic (0.5%) and higher (5%) concentrations of chemically enhanced advanced-primary (PL) and full-secondary treated (HTP) effluents from two southern California wastewater treatment plants (WWTP). Hepatic gene expression was examined using a custom low-density microarray. <br><br>
Project description:Many biomonitoring tools/approaches have been proposed to assess presence of endocrine active chemicals (EACs) and their biological effects in the field. Although these tools have provided valuable information, they are often limited by their specificity for certain groups of EACs and they may not account for interactions between EACs. This study aims to evaluate utility of transcriptomic and metabolomic technologies for effects monitoring in the field, and to advance integration of omic and environmental chemistry data sets. The objective was to utilize transcriptomic biomonitoring to determine the relative contribution of wastewater treatment plant effluents to biological effects observed in fish exposed to ambient waters receiving the effluents. Adult male fathead minnow were exposed to treated wastewater effluent or stream water up or downstream the plant in three different watersheds for 4 days. After exposure, the liver of 5-7 fish per treatment per site (i.e 19-21 fish from each watershed) were analyzed by microarrays. The transcriptomic profiles were compared to control fish exposed to Lake Superior filtered water.
Project description:There is an increasing drive to replace fish oil (FO) in finfish aquaculture diets with vegetable oils (VO), driven by the short supply of FO derived from wild fish stocks. Little is known of the consequences for fish health after such substitution. The effect of dietary VO on hepatic gene expression was determined in Atlantic salmon (Salmo salar) byg a cDNA microarray analysis. Post-smolt farmed salmon were reared for x weeks on diets where the FO component of the feed was replaced with one of three different VOs - rapeseed (RO), soybean (SO) or linseed (LO). RNA from five fish fed on each diet was extracted. A total of 20 cDNA microarray hybridisations - TRAITS / SGP Atlantic salmon 17k feature cDNA microarray - were performed - 4 diets (three VO + FO control) x 5 individuals - using a common pooled reference control design. Data were obtained from 19 of the 20 hybridisations.