Unexpected regulatory functions of cyprinid Viperin on inflammation and metabolism.

BACKGROUND: Viperin, also known as radical S-adenosyl-methionine domain containing protein 2 (RSAD2), is an interferon-inducible protein that is involved in the innate immune response against a wide array of viruses. In mammals, Viperin exerts its antiviral function through enzymatic conversion of cytidine triphosphate (CTP) into its antiviral analog ddhCTP as well as through interactions with host proteins involved in innate immune signaling and in metabolic pathways exploited by viruses during their life cycle. However, how Viperin modulates the antiviral response in fish remains largely unknown. RESULTS: For this purpose, we developed a fathead minnow (Pimephales promelas) clonal cell line in which the unique viperin gene has been knocked out by CRISPR/Cas9 genome-editing. In order to decipher the contribution of fish Viperin to the antiviral response and its regulatory role beyond the scope of the innate immune response, we performed a comparative RNA-seq analysis of viperin-/- and wildtype cell lines upon stimulation with recombinant fathead minnow type I interferon. CONCLUSIONS: Our results revealed that Viperin does not exert positive feedback on the canonical type I IFN but acts as a negative regulator of the inflammatory response by downregulating specific pro-inflammatory genes and upregulating repressors of the NF-κB pathway. It also appeared to play a role in regulating metabolic processes, including one carbon metabolism, bone formation, extracellular matrix organization and cell adhesion.

Toxicogenomics of Five Cytostatics in Fathead Minnow (Pimephales promelas) Larvae.

In this study, the toxicogenomic effects of five cytostatics (tamoxifen, methotrexate, capecitabine, cyclophosphamide, and ifosfamide) on fathead minnow (Pimephales promelas) larvae were evaluated. Post-fertilization eggs were exposed to increasing concentrations of the drugs for six days. The expression levels of two genetic biomarkers for toxicity and four thyroid hormone-related gene pathways were measured. Interestingly, the results showed that all concentrations of the five cytostatics affect the transcription levels of both toxicity biomarker genes. Additionally, the thyroid hormone-related genes had different expression levels than the control, with the most significant changes observed in those larvae exposed to cyclophosphamide and ifosfamide. While a previous study found no effects on fish morphology, this study suggests that the five cytostatics modify subtle molecular responses of P. promelas, highlighting the importance of assessing multibiological level endpoints throughout the lifecycle of animals to understand the full portrait of potential effects of cytostatics and other contaminants.

Uptake of Per- and Polyfluoroalkyl Substances by Fish, Mussel, and Passive Samplers in Mobile-Laboratory Exposures Using Groundwater from a Contamination Plume at a Historical Fire Training Area, Cape Cod, Massachusetts.

Aqueous film-forming foams historically were used during fire training activities on Joint Base Cape Cod, Massachusetts, and created an extensive per- and polyfluoroalkyl substances (PFAS) groundwater contamination plume. The potential for PFAS bioconcentration from exposure to the contaminated groundwater, which discharges to surface water bodies, was assessed with mobile-laboratory experiments using groundwater from the contamination plume and a nearby reference location. The on-site continuous-flow 21-day exposures used male and female fathead minnows, freshwater mussels, polar organic chemical integrative samplers (POCIS), and polyethylene tube samplers (PETS) to evaluate biotic and abiotic uptake. The composition of the PFAS-contaminated groundwater was complex and 9 PFAS were detected in the reference groundwater and 17 PFAS were detected in the contaminated groundwater. The summed PFAS concentrations ranged from 120 to 140 ng L-1 in reference groundwater and 6100 to 15,000 ng L-1 in contaminated groundwater. Biotic concentration factors (CFb) for individual PFAS were species, sex, source, and compound-specific and ranged from 2.9 to 1000 L kg-1 in whole-body male fish exposed to contaminated groundwater for 21 days. The fish and mussel CFb generally increased with increasing fluorocarbon chain length and were greater for sulfonates than for carboxylates. The exception was perfluorohexane sulfonate, which deviated from the linear trend and had a 10-fold difference in CFb between sites, possibly because of biotransformation of precursors such as perfluorohexane sulfonamide. Uptake for most PFAS in male fish was linear over time, whereas female fish had bilinear uptake indicated by an initial increase in tissue concentrations followed by a decrease. Uptake of PFAS was less for mussels (maximum CFb = 200) than for fish, and mussel uptake of most PFAS also was bilinear. Although abiotic concentration factors were greater than CFb, and values for POCIS were greater than for PETS, passive samplers were useful for assessing PFAS that potentially bioconcentrate in fish but are present at concentrations below method quantitation limits in water. Passive samplers also accumulate short-chain PFAS that are not bioconcentrated.

Efficacy of hydrogen peroxide to reduce Gyrodactylus species infestation density on four fish species.

OBJECTIVE: The ability to effectively treat parasitic infestations of fish is of high importance for fish culture facilities. However, tools or approved therapies for treating infestations on fish are limited. This paper summarizes results from four separate clinical field studies that evaluated the efficacy of hydrogen peroxide (H2 O2 ; 35% PEROX-AID) for reducing Gyrodactylus spp. infestation density. METHODS: Three species of Gyrodactylus were studied (G. salmonis, hosts: Brook Trout Salvelinus fontinalis and Lake Trout S. namaycush; G. freemani, host: Yellow Perch Perca flavescens; G. hoffmani, host: Fathead Minnow Pimephales promelas) before and after the application of immersion H2 O2 therapy. RESULT: Parasite density was significantly reduced for each parasite × host combination to which H2 O2 therapy was applied. Two clinical field studies in salmonids were found to demonstrate substantial effectiveness that enabled 35% PEROX-AID approval. CONCLUSION: Further assessments of Gyrodactylus spp. could expand the use of H2 O2 for controlling these parasites in aquaculture. Specifically, H2 O2 was effective at all levels tested (50 or 75 mg H2 O2 /L for 60 min for the Yellow Perch and Fathead Minnow clinical field studies; 100 or 150 mg H2 O2 /L for 30 min regardless of salt pre-treatment for the Brook Trout study; and 100 mg H2 O2 /L for 30 min or 50 mg H2 O2 /L for 60 min for the Lake Trout study).

Isolation and characterization of plasma-derived exosomes from olive flounder (Paralichthys olivaceus) and their wound healing and regeneration activities.

Exosomes have garnered enormous interest for their role in physiological and pathological processes and their potential for therapeutic and diagnostic applications. In this study, exosomes were isolated from plasma of olive flounder (Paralichthys olivaceus) and their physiochemical and morphological characteristics, as well as wound healing and regeneration activities were determined. Isolated exosomes had typical characteristics, including average particle diameter (151.82 ± 9.17 nm), concentration (6.31 × 1010 particles/mL) with a membrane-bound, cup-shaped morphology. Exosome marker proteins, tetraspanins (CD63, CD9, and CD81), and acetylcholinesterase were detected, indicating the presence of exosomes in olive flounder plasma. Exosomes exhibited no toxicity in in vitro and in vivo studies, even at the highest treatment concentrations (100 and 400 µg/mL, respectively), confirming their suitability for further functional studies. Following exosome treatment (50 and 100 µg/mL), substantial cell migration with rapid closure of the open wound area in in vitro scratch wound healing assay and faster zebrafish larvae fin regeneration rate was observed compared to that of the vehicle. Moreover, exosomes exhibited immunomodulatory properties associated with wound healing, based on mRNA expression patterns in fathead minnow (FHM) cells. In conclusion, exosomes isolated from olive flounder plasma using ultracentrifugation exhibited minimal toxicity and enhanced wound healing and tissue regeneration activities. Identification and in-depth investigation of olive flounder plasma-derived exosome constituents will support the development of exosomes as an efficient therapeutic carrier system for fish medicine in the future.

Physical Evidence of Oil Uptake and Toxicity Assessment of Amphiphilic Grafted Nanoparticles Used as Oil Dispersants.

Herein, we report the toxicity evaluation of a new prototype dispersant system, silicon dioxide nanoparticles (NPs) functionalized with (3-glycidoxypropyl)triethoxysilane (GPS) and grafted poly(ε-caprolactone)-block-poly[oligo(ethylene glycol)methyl methacrylate mono-methyl ether] (NP-PCL-POEGMA). This serves as a follow up of our previous study where grafted silicon dioxide NPs functionalized with GPS and grafted hyperbranched poly(glycidol) (NP-HPG) were evaluated for reducing the toxicity in embryo, juvenile, and adult fish populations. In this study, the NP-HPG sample is used as a baseline to compare against the new NP-PCL-POEGMA samples. The relative size was established for three NP-PCL-POEGMA samples via cryogenic transmission electron microscopy. A quantitative mortality study determined that these NPs are non-toxic to embryo populations. An ethoxyresorufin-O-deethylase assay was performed on these NP-PCL-POEGMA samples to test for reduced cytochrome P450 1A after the embryos were exposed to the water-accommodated fraction of crude oil. Overall, these NP-PCL-POEGMA NPs better protected the embryo populations than the previous NP-HPG sample (using a protein activity end point), showing a trend in the right direction for prototype dispersants to replace the commercially utilized Corexit.

DEVELOPING INTEGRAL PROJECTION MODELS FOR ECOTOXICOLOGY.

In many ecosystems, especially aquatic ecosystems, size plays a critical role in the factors that determine an individual's ability to survive and reproduce. In aquatic ecotoxicology, size informs both realized and potential acute and chronic effects of chemical exposure. This paper demonstrates how chemical and nonchemical effects on growth, survival, and reproduction can be linked to population-level dynamics using size-structured integral projection models (IPM). The modeling approach was developed with the goals and constraints of ecological risk assessors in mind, who are tasked with estimating the effects of chemical exposures to wildlife populations in a data-limited environment. The included case study is a collection of daily time-step IPMs parameterized for the life history and annual cycle of fathead minnows (Pimephales promelas), which motivated the development of modeling techniques for seasonal, iteroparous reproduction, density dependent growth effects, and size-dependent over-winter survival. The effects of a time-variable annual chemical exposure were interpreted using a toxicokinetic-toxicodynamic model for acute survival and sub-lethal growth effects model for chronic effects and incorporated into the IPMs. This paper presents a first application of integral projection models to ecotoxicology. Our research demonstrates that size-structured IPMs provide a promising, flexible, framework for synthesizing ecotoxicologically relevant data and theory to explore the effects of chemical and nonchemical stressors and the resulting impacts on exposed populations.

Development of a Comprehensive Toxicity Pathway Model for 17α-Ethinylestradiol in Early Life Stage Fathead Minnows (Pimephales promelas).

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.

Investigating the Predictive Power of Three Potential Sublethal Endpoints for the Fathead Minnow Fish Embryo Toxicity Test: Snout-Vent Length, Eye Size, and Pericardial Edema.

The fish embryo acute toxicity (FET) test is known to be less sensitive than the fish acute test for some chemicals, including neurotoxicants. Thus, there is an interest in identifying additional endpoints that can improve FET test performance. The goal of this project was to advance alternative toxicity testing methods by determining whether select developmental abnormalities-snout-vent length, eye size, and pericardial area-are linked to adverse alterations in ecologically-relevant behaviors and delayed mortality. Fathead minnow (Pimephales promelas) FET tests were conducted with 3,4-dicholoroaniline, cadmium, and perfluorooctanesulfonic acid (PFOS) and developmental abnormalities were quantified. Surviving eleutheroembryos were reared in clean water to 14 days post fertilization (dpf), during which time behaviors and mortality were evaluated. None of the abnormalities evaluated were predictive of behavioral alterations; however, embryos with ≥14% reductions in length or ≥3.54-fold increases in pericardial area had an 80% chance of mortality by 14 dpf. When these abnormalities were used as markers of mortality, the LC50s for cadmium and PFOS were less than those calculated using only standardized FET test endpoints and similar to those obtained via larval fish tests, indicating that the snout-vent length and pericardial area warrant consideration as standard FET test endpoints.

Impact of feed restriction, chloroquine and deoxynivalenol on viral haemorrhagic septicaemia virus IVb in fathead minnow Pimephales promelas Rafinesque.

Autophagy can markedly alter host response to infectious disease, and several studies have demonstrated that a restricted diet or deoxynivalenol modulates autophagy and reduces mortality of fish due to bacterial disease. The picture is less clear for viral diseases of fish. Duplicate tanks of fathead minnow, Pimephales promelas Rafinesque, were fed a replete diet (control), 100 µM chloroquine, 5 µM deoxynivalenol, 10% (fasted) or 40% of a replete diet (pair-fed) for 2 weeks and then experimentally infected by intraperitoneal injection with 2 × 105 viral haemorrhagic septicaemia virus IVb. Survival from highest to lowest for the different treatments was as follows: deoxynivalenol (average 43.3%); control (40.0%); pair-fed (35.0%); fasted (33.3%); and chloroquine (21.7%). No treatment significantly altered the survival rate of fathead minnow after VHSV IVb infection when compared to controls; however, the fish fed with chloroquine had significantly lower survival rate than the fish fed deoxynivalenol (p < .05).

Advancing the fathead minnow (Pimephales promelas) as a model for immunotoxicity testing: Characterization of the renal transcriptome following Yersinia ruckeri infection.

Recent studies have utilized the fathead minnow (Pimephales promelas) to explore the immunotoxic effects associated with a variety of environmental contaminants in the absence of immunological stimuli. Though this approach allows for alterations in the resting immune system to be detected, previous evidence suggests that many immunotoxic effects may only manifest in the activated immune system. However, basic immune responses to pathogens have not been well described in this species. To expand the utility of the fathead minnow as a model for immunotoxicity testing, a more comprehensive understanding of the activated immune system is required. As such, the main goal of this study was to characterize the transcriptomic response to pathogen infection in the fathead minnow using RNA sequencing. To achieve this goal, female fathead minnows were intraperitoneally injected with either Hank's Balanced Salt Solution (sham-injected) or Yersinia ruckeri (pathogen-injected). Eight hours following injection, fish were sacrificed for the assessment of general morphological (i.e., mass, length, condition factor, hepatic index) and immunological (i.e., leukocyte counts, spleen index) endpoints. To assess the molecular immune response to Y. ruckeri, kidney tissue was collected for transcriptomic analysis. A comparison of sham- and pathogen-injected fish revealed that >1800 genes and >500 gene networks were differentially expressed.Gene networks associated with inflammation, innate immunity, complement, hemorrhaging and iron absorption are highlighted and their utility within the context of immunotoxicity is discussed. These data reveal pathogen-related molecular endpoints to improve data interpretation of future studies utilizing the fathead minnow as a model for immunotoxicity.

Identification of the Nrf2 in the fathead minnow muscle cell line: role for a regulation in response to H2O2 induced the oxidative stress in fish cell.

The Nrf2 (nuclear factor erythroid 2-related factor 2) plays a central role in cell protection against a wide variety of environmental stressors through the Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway, but its involvement in modulation of antioxidant system of fish cell is still largely unexplored. The present study focused on the molecular cloning and silencing of the Nrf2 in the fathead minnow muscle cell line (FHM) in response to the oxidative stress induced by H2O2. A full-length cDNA of coding Nrf2 was cloned from FHM cells by RT-PCR and RACE approaches. The obtained cDNA covered 2578 bp with an open reading frame (1770 bp) of encoding 589 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (51-86%) among 16 fishes. Based on the cloned Nrf2 sequence, the siRNA-242 of targeting Nrf2 with the best knocking down efficiency was designed and detected. Then, the mRNA levels of Keap1, Nrf2, Maf (musculoaponeurotic fibrosarcoma oncogene), and HO-1 (haemoxygenase-1); the activities of T-SOD (total superoxide dismutase), CAT (catalase), and GSH-PX (glutathione peroxidase); the levels of GSH (glutathione) and MDA (malonaldehyde); and the cell cycle and apoptosis were analyzed to investigate the molecular responses after H2O2 exposure. These results showed a coordinated transcriptional regulation of Keap1, Maf, and HO-1 and antioxidants (T-SOD, GSH, CAT, and GSH-PX) and MDA levels after H2O2 exposure, leading to oxidative damage and apoptosis. These findings provided an insight to understand the mechanisms of Nrf2 against oxidative stress in fish.

Effects of Environmentally Relevant Residual Levels of Diluted Bitumen on Wild Fathead Minnows (Pimephales promelas).

Transportation of crude oil across North America's boreal ecozone creates the potential for spills in freshwater where less is known about the sensitivity of resident fish than for marine systems. The sensitivity of wild fathead minnows (FHM) to residual concentrations (ppb range) of the water accommodated fraction (WAF) of diluted bitumen (dilbit) was assessed by exposing them for 21 days followed by a 14 days depuration. Target concentrations were well below detection limits for GC-MS, but were estimated by dilution factor (1:100,000 and 1:1,000,000 WAF:water) to contain less than 0.0003 µg/L of polycyclic aromatic compounds. Confinement and handling stress caused by transfer of wild fish into tanks much smaller than their natural range resulted in mortality and lower body condition among all groups, but interactive effects of oil exposures still resulted in females with smaller cortical alveolar oocytes, and males with larger testicular lobe lumen sizes. Additional studies examining the compounded effects of stress and environmentally relevant oil exposures in wild fishes are needed.

Evidence for incipient alarm signalling in fish.

In Focus: Bairos-Novak, K.R., Ferrari, M.C.O., & Chivers, D.P. (2019). A novel alarm signal in aquatic prey: Familiar minnows coordinate group defences against predators through chemical disturbance cues. Journal of Animal Ecology, 88, 1281-1290, https://doi.org/10.1111/1365-2656.12986. Chemicals released during predation have long been studied as cues to nearby prey that predators are active in the vicinity. Until now, these chemicals have been labelled as cues because there was no compelling evidence for the necessary components of a communication system, namely (a) voluntary control of release of information, (b) capacity for graded responses and (c) the presence of specialized structures for the production and release of the signal. New findings by Bairos-Novak, Ferrari, and Chivers (2019) show that fathead minnows alter the potency of disturbance "cues" when in the presence of other fathead minnows compared to when they are alone and produce either more or different disturbance "cues" when in the presence of familiar conspecifics compared to when they are in the presence of unfamiliar conspecifics. The behavioural response to these cues is shoaling, which would confer fitness benefits to the sender, thereby qualifying as a signal rather than a cue. This is a significant advancement in the field of chemical ecology of aquatic organisms because disturbance "cues" by fathead minnows bear two of the three hallmarks of an incipient disturbance "signal".

A novel alarm signal in aquatic prey: Familiar minnows coordinate group defences against predators through chemical disturbance cues.

Animal signalling systems outside the realm of human perception remain largely understudied. These systems consist of four main components: a signalling context, a voluntary signal, receiver responses and resulting fitness benefits to both the signaller and receiver(s). It is often most difficult to determine incidental cues from voluntary signals. One example is chemical disturbance cues released by aquatic prey during predator encounters that may serve to alert conspecifics of nearby risk and initiate tighter shoaling. We aimed to test whether disturbance cues are released incidentally (i.e. as a cue) or are produced voluntarily depending on a specific signalling context such as the audience surrounding the individual, and thus constitute a signal. We hypothesized that if receivers use disturbance cues to communicate risk among themselves, they would produce more (or more potent) disturbance cues when present in a group of conspecifics rather than when they are isolated (presence/absence of an audience) and use disturbance cues more when present alongside familiar rather than unfamiliar conspecifics (audience composition effect). We placed fathead minnows (Pimephales promelas) in groups with familiar fish, unfamiliar fish or as isolated individuals with no audience present, and then simulated a predator chase to evoke disturbance cues. We used bioassays with independent receivers to assess whether the disturbance cues produced differed depending on the signallers' audience. We found evidence of voluntary signalling, as minnows responded to disturbance cues from groups of fish with tighter shoaling while disturbance cues from isolated minnows did not evoke a significant shoaling response (presence/absence audience effect). Receivers also increased shoaling, freezing and dashing more in response to disturbance cues from familiar groups compared to disturbance cues from unfamiliar groups or isolated minnows (audience composition effect). Together, these findings support our hypothesis that disturbance cues are used as an antipredator signal to initiate coordinated group defences among familiar conspecifics involving shoaling, freezing and dashing. This study represents the strongest evidence to date that chemicals released by aquatic prey upon disturbance by predators serve as voluntary signals rather than simply cues that prey have evolved to detect when assessing their risk of predation.

In vitro transcribed dsRNA limits viral hemorrhagic septicemia virus (VHSV)-IVb infection in a novel fathead minnow (Pimephales promelas) skin cell line.

The farming of baitfish, fish used by anglers to catch predatory species, is of economic and ecological importance in North America. Baitfish, including the fathead minnow (Pimephales promelas), are susceptible to infection from aquatic viruses, such as viral hemorrhagic septicemia virus (VHSV). VHSV infections can cause mass mortality events and have the potential to be spread to novel water bodies through baitfish as a vector. In this study, a novel skin cell line derived from fathead minnow (FHMskin) is described and its use as a tool to study innate antiviral immune responses and possible therapies is introduced. FHMskin grows optimally in 10% fetal bovine serum and at warmer temperatures, 25-30 °C. FHMskin is susceptible and permissive to VHSV-IVb infection, producing high viral titres of 7.35 × 107 TCID50/mL after only 2 days. FHMskin cells do not experience significant dsRNA-induced death after treatment with 50-500 ng/mL of in vitro transcribed dsRNA for 48 h and respond to dsRNA treatment by expressing high levels of three innate immune genes, viperin, ISG15, and Mx1. Pretreatment with dsRNA for 24 h significantly protected cells from VHSV-induced cell death, 500 ng/mL of dsRNA reduced cell death from 70% to less than 15% at a multiplicity of infection of 0.1. Thus, the novel cell line, FHMskin, represents a new method for producing high tires of VHSV-IVb in culture, and for studying dsRNA-induced innate antiviral responses, with future applications in dsRNA-based antiviral therapeutics.

Control performance of fish short term reproduction assays with fathead minnow (Pimephales promelas).

The fish short-term reproduction assay (FSTRA) is an in vivo screen to assess potential interactions with the fish endocrine system. After a 21-day exposure period vitellogenin (VTG) and secondary sexual characteristics are measured in males and females. Egg production and fertility are also monitored daily throughout the test. This paper presents data from 49 studies performed to satisfy test orders from the United States Environmental Protection Agency's Endocrine Disruptor Screening Program. Data Evaluation Records were used to collate the typical control variability and performance of test parameters in FSTRAs conducted in different laboratories with fathead minnow (Pimephales promelas). We also examine the statistical power of FSTRA endpoints and assess whether available historical control data (HCD) assist evidence-based interpretation of the endpoints. Statistically significant inter-laboratory differences were found for all endpoints except survival. HCD could therefore be usefully developed on a laboratory-by-laboratory basis to aid interpretation of new study data. Reliable HCD ranges could be developed for survival, body weight/length, gonadal somatic index, fertilisation success, and male tubercle score, and used in association with stated test acceptability criteria to interpret FSTRA data. In contrast, high intra- and inter-laboratory control variability for VTG and fecundity means that HCD for these endpoints are of limited use during study interpretation.

Effects of selenium on benthic macroinvertebrates and fathead minnow (Pimephales promelas) in a boreal lake ecosystem.

Selenium (Se) is a contaminant of concern in many aquatic ecosystems due to its narrow range between essentiality and toxicity in oviparous (yolk-bearing) vertebrates. The objective of the present study was to determine the effects of Se, experimentally added to in situ limnocorrals as selenite, on invertebrate communities and fathead minnow (Pimephales promelas) at environmentally realistic Se concentrations. Nine limnocorrals were deployed in a mesotrophic lake at the International Institute for Sustainable Development - Experimental Lakes Area in Ontario, Canada in May 2017. From June 1 to August 17, 2017, selenite was added to six enclosures to attain mean measured aqueous Se concentrations of 1.0 ±â€¯0.10 or 8.9 ±â€¯2.7 µg/L Se (in triplicate) and three limnocorrals were untreated controls (background mean aqueous Se = 0.12 ±â€¯0.03 µg/L). Benthic macroinvertebrates were collected throughout and at the end of the exposure period using artificial substrates to determine density, dry biomass, diversity, and taxa richness at the family level. Reproductively mature female fathead minnows (added on d 33 of the study) were collected throughout and at the end of the exposure period. After 77 d, Chironomidae and Gammaridae densities and biomass were significantly lower in the 8.9 µg/L Se treatment relative to the 1.0 µg/L Se treatment and the control. Invertebrate diversity (measured as Shannon's and Simpson's indices) significantly declined in the 1.0 µg/L and 8.9 µg/L Se treatments relative to the control (0.12 µg/L Se group). Fulton's condition factor for fathead minnow was significantly less in the 8.9 µg/L treatment compared to 0.12 and 1.0 µg/L Se experimental groups. The results of this study indicated that exposure to relatively low aqueous selenite concentrations can negatively affect invertebrate density and biomass, as well as fish condition. More research is necessary to characterize the risk of selenite exposure to aquatic invertebrates under realistic field conditions, and future risk assessments may need to consider reduced food availability as a factor that may impair the health of higher trophic level organisms in areas with elevated selenite.

Embryo-larval BDE-47 exposure causes decreased pathogen resistance in adult male fathead minnows (Pimephales promelas).

Exposures to polybrominated diphenyl ethers (PBDEs) have been shown to alter immune function in adult organisms across a variety of taxa. However, few if any studies have investigated the long-term consequences of early life stage PBDE exposures on immune function in fish. This study sought to determine the effects of early life stage BDE-47 exposure on pathogen resistance in the fathead minnow (Pimephales promelas) following an extended depuration period (≥180 d). Minnows were exposed to BDE-47 via a combination of maternal transfer and diet through 34 days post fertilization (dpf), raised to adulthood (>215 dpf) on a clean diet, then subjected to pathogen resistance trials. Early life stage exposures to BDE-47 did not affect the ability of females to survive from Yersinia ruckeri infection. However, the survival of BDE-47 exposed males was significantly reduced relative to controls, indicating that developmental exposures to BDE-47 altered male immunity. Because BDE-47 is a known thyroid hormone disruptor and thyroid hormone disruptors have the potential to adversely impact immune development and function, metrics indicative of thyroid disruption were evaluated, as were immune parameters known to be altered in response to thyroid disruption. BDE-47 exposed minnows exhibited signs of thyroid disruption (i.e., reduced growth); however, no alterations were observed in immune parameters known to be influenced by thyroid hormones (i.e., thymus size, expression of genes associated with lymphoid development) suggesting that the observed alterations in immunocompetence may occur through alternative mechanisms. Regardless of the mechanisms responsible, the results of this study demonstrate the potential for early life stage PBDE exposures to adversely impact immunity and illustrate that the immunological consequences of PBDE exposures are sex dependent.

Estrogen signaling through both membrane and nuclear receptors in the liver of fathead minnow.

Estradiol is a potent sex steroid hormone that controls reproduction and other cellular pathways in fish. It is known to regulate important proteins such as vitellogenin, the egg yolk precursor protein, and zona radiata proteins that form the eggshell for fish eggs. These proteins are made in the liver and transported out into the blood from where they are taken up into the ovary during oogenesis. Estradiol can exert its influence directly through soluble nuclear receptors (there are three in fish) or indirectly through membrane receptors and a phosphorylation cascade. Often there is coordination through both genomic and non-genomic pathways. We have used a toxicogenomics approach to determine the contribution of genomic and non-genomic regulation in the liver of fathead minnows exposed to 5ng ethinylestradiol per liter or to a mixture of 5ng ethinylestradiol and 100ng ZM189,154 (ZM) per liter. ZM has previously been shown to be a "perfect" antagonist for the fish nuclear estrogen receptors but has displayed agonistic activities for membrane receptors. We find that both nuclear and membrane receptors contribute to the biosynthesis of vitellogenin 1 and estrogen receptor one (Esr1), among others. In addition, lipid metabolism pathways appear to require both activities.