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.

Impacts of caffeine on fathead minnow behaviour and physiology.

Pollution from regularly used substances such as pharmaceuticals, cleaning agents, and even food and beverages is an increasing problem in the environment. Caffeine, a commonly ingested stimulant, is one such contaminant that has been detected in aquatic environments worldwide. Yet, little is known about how ecologically relevant concentrations of caffeine influence the morphology, behaviour, and physiology of exposed organisms. To address this knowledge gap, we exposed fathead minnow (Pimephales promelas) to three caffeine treatments: a freshwater control (nominal: 0 ng/L), a low (nominal: 1,000 ng/L) and high environmentally relevant dose (nominal: 10,000 ng/L), for 35 days. We tested the learning abilities, anxiety, metabolic rates, and morphological features of exposed vs. control fish. Caffeine exposure did not affect the ability of fish to learn but did influence anxiety levels. Over the course of repeated anxiety testing, unexposed control fish visited a black square more often while fish exposed to low levels of caffeine did not, potentially indicating that these fish remained in a more anxious state. While caffeine did not impact metabolism, fish growth, or body size, it was associated with lower liver investment-although this response was only observed in our low caffeine treatment. Overall, our results suggest that even relatively low concentrations of caffeine may impact the liver size and anxiety of exposed fish, but further research is needed to assess how extended exposure to caffeine impacts fitness. Given the increase in anthropogenic contaminants in aquatic environments, it is important that we continue to investigate their effects on the organisms exposed to them.

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.

Proteome changes in larval zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) exposed to (±) anatoxin-a.

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following  (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01-3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding.

Effects of Age and Exposure Duration on the Sensitivity of Early Life Stage Fathead Minnow (Pimephales promelas) to Waterborne Propranolol Exposure.

Propranolol is a heavily prescribed, nonspecific beta-adrenoceptor (bAR) antagonist frequently found in wastewater effluents, prompting concern over its potential to adversely affect exposed organisms. In the present study, the transcriptional responses of 4, 5, and 6 days postfertilization (dpf) ±1 h fathead minnow, exposed for 6, 24, or 48 h to 0.66 or 3.3 mg/L (nominal) propranolol were characterized using RNA sequencing. The number of differentially expressed genes (DEGs) was used as an estimate of sensitivity. A trend toward increased sensitivity with age was observed; fish >7 dpf at the end of exposure were particularly sensitive to propranolol. The DEGs largely overlapped among treatment groups, suggesting a highly consistent response that was independent of age. Cluster analysis was performed using normalized count data for unexposed and propranolol-exposed fish. Control fish clustered tightly by age, with fish ≥7 dpf clustering away from younger fish, reflecting developmental differences. When clustering was conducted using exposed fish, in cases where propranolol induced a minimal or no transcriptional response, the results mirrored those of the control fish and did not appreciably cluster by treatment. In treatment groups that displayed a more robust transcriptional response, the effects of propranolol were evident; however, fish <7 dpf clustered away from older fish, despite having similar numbers of DEGs. Increased sensitivity at 7 dpf coincided with developmental milestones with the potential to alter propranolol pharmacokinetics or pharmacodynamics, such as the onset of exogenous feeding and gill functionality as well as increased systemic expression of bAR. These results may have broader implications because toxicity testing often utilizes fish <4 dpf, prior to the onset of these potentially important developmental milestones, which may result in an underestimation of risk for some chemicals. Environ Toxicol Chem 2024;43:807-820. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Short-Term Transcriptomic Points of Departure Are Consistent with Chronic Points of Departure for Three Organophosphate Pesticides across Mouse and Fathead Minnow.

New approach methods (NAMs) can reduce the need for chronic animal studies. Here, we apply benchmark dose (concentration) (BMD(C))-response modeling to transcriptomic changes in the liver of mice and in fathead minnow larvae after short-term exposures (7 days and 1 day, respectively) to several dose/concentrations of three organophosphate pesticides (OPPs): fenthion, methidathion, and parathion. The mouse liver transcriptional points of departure (TPODs) for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-bw/day, while the fathead minnow larva TPODs were 0.007, 0.115, and 0.046 mg/L, respectively. The TPODs were consistent across both species and reflected the relative potencies from traditional chronic toxicity studies with fenthion identified as the most potent. Moreover, the mouse liver TPODs were more sensitive than or within a 10-fold difference from the chronic apical points of departure (APODs) for mammals, while the fathead minnow larva TPODs were within an 18-fold difference from the chronic APODs for fish species. Short-term exposure to OPPs significantly impacted acetylcholinesterase mRNA abundance (FDR p-value <0.05, |fold change| ≥2) and canonical pathways (IPA, p-value <0.05) associated with organism death and neurological/immune dysfunctions, indicating the conservation of key events related to OPP toxicity. Together, these results build confidence in using short-term, molecular-based assays for the characterization of chemical toxicity and risk, thereby reducing reliance on chronic animal studies.

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).

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.

Maternal Transfer and Apical and Physiological Effects of Dietary Hexabromocyclododecane Exposure in Parental Fathead Minnows (Pimephales promelas).

Hexabromocyclododecane (HBCD) is a persistent organic pollutant that has been characterized as an endocrine disruptor, undergoes maternal transfer, and hinders development and growth in oviparous organisms. The present study examined the apical effects of dietary HBCD (11.5, 36.4, 106 mg/kg, wet wt) on adult fathead minnow exposed for 49 days and the subsequent accumulation and maternal transfer kinetics in adult tissue and eggs, respectively. Exposed adults displayed a significant increase in egg production in the medium treatment group, but no other significant effects were noted. Maternal transfer of dietary HBCD had a similar egg-to-muscle ratios (EMR) in the low and medium treatment groups (1.65 and 1.27 [wet wt], respectively). However, the high treatment group deviated from other treatments with an EMR of 4.2 (wet wt), potentially due to differences in total lipid content in food and/or reaching diffusion/lipid saturation limits in adult tissue, resulting in lower accumulation in the adult muscle tissue. A positive correlation was observed between egg HBCD concentration and time of exposure, which indicates that maternal transfer of HBCD is of concern in fish, and further studies should be conducted to fully elucidate the potential adverse effects that may be observed in the early life stage of oviparous organisms. Environ Toxicol Chem 2023;42:143-153. © 2022 SETAC.

Genome resequencing clarifies phylogeny and reveals patterns of selection in the toxicogenomics model Pimephales promelas.

Background: The fathead minnow (Pimephales promelas) is a model species for toxicological research. A high-quality genome reference sequence is available, and genomic methods are increasingly used in toxicological studies of the species. However, phylogenetic relationships within the genus remain incompletely known and little population-genomic data are available for fathead minnow despite the potential effects of genetic background on toxicological responses. On the other hand, a wealth of extant samples is stored in museum collections that in principle allow fine-scale analysis of contemporary and historical genetic variation. Methods: Here we use short-read shotgun resequencing to investigate sequence variation among and within Pimephales species. At the genus level, our objectives were to resolve phylogenetic relationships and identify genes with signatures of positive diversifying selection. At the species level, our objective was to evaluate the utility of archived-sample resequencing for detecting selective sweeps within fathead minnow, applied to a population introduced to the San Juan River of the southwestern United States sometime prior to 1950. Results: We recovered well-supported but discordant phylogenetic topologies for nuclear and mitochondrial sequences that we hypothesize arose from mitochondrial transfer among species. The nuclear tree supported bluntnose minnow (P. notatus) as sister to fathead minnow, with the slim minnow (P. tenellus) and bullhead minnow (P. vigilax) more closely related to each other. Using multiple methods, we identified 11 genes that have diversified under positive selection within the genus. Within the San Juan River population, we identified selective-sweep regions overlapping several sets of related genes, including both genes that encode the giant sarcomere protein titin and the two genes encoding the MTORC1 complex, a key metabolic regulator. We also observed elevated polymorphism and reduced differentation among populations (FST) in genomic regions containing certain immune-gene clusters, similar to what has been reported in other taxa. Collectively, our data clarify evolutionary relationships and selective pressures within the genus and establish museum archives as a fruitful resource for characterizing genomic variation. We anticipate that large-scale resequencing will enable the detection of genetic variants associated with environmental toxicants such as heavy metals, high salinity, estrogens, and agrichemicals, which could be exploited as efficient biomarkers of exposure in natural populations.

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.

Acute Toxicity of Major Geochemical Ions to Fathead Minnows (Pimephales promelas): Part B-Modeling Ion Toxicity.

Mathematical models are presented for the acute median lethal concentrations of major geochemical ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO4 2- , HCO3 - /CO3 2- ) to fathead minnows (Pimephales promelas), based on an extensive series of experiments presented in a companion article. Toxicity relationships across different dilution waters, individual salts, and salt mixtures suggest six independent mechanisms of toxicity to consider in modeling efforts, including Mg/Ca-specific toxicity, osmolarity-related toxicity, SO4 -specific toxicity, K-specific toxicity, effects of high pH/alkalinity, and a multiple ion-related toxicity at low Ca distinct from the other mechanisms. Models are evaluated using chemical activity-based exposure metrics pertinent to each mechanism, but concentration-based alternative models that are simpler to apply are also addressed. These models are compared to those previously provided for Ceriodaphnia dubia, and various issues regarding their application to risk assessments are discussed. Environ Toxicol Chem 2022;41:2095-2106. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

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.

Acute Toxicity of Major Geochemical Ions to Fathead Minnows (Pimephales Promelas): Part A-Observed Relationships for Individual Salts and Salt Mixtures.

The results of a series of experiments on the acute toxicity of major geochemical ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO4 2- , HCO3 - /CO3 2- ) to fathead minnows (Pimephales promelas) are reported. Tests of individual major ion salts in various dilution waters demonstrated that the toxicities of Na, Mg, and K salts decrease as the overall ion content of the dilution water increases. For Na and Mg salts, this is attributable to Ca content as previously reported for Ceriodaphnia dubia. For K salts, the cause is unclear, but it is not due to Na as reported for C. dubia. In an unregulated test at high pH (9.3), NaHCO3 was also found to be twice as toxic compared to when the pH was reduced to 8.4. Experiments with binary salt mixtures indicated the existence of multiple independent mechanisms of action. These include K-specific toxicity and Ca/Mg-specific toxicity previously reported for C. dubia, but also apparent toxicities related to SO4 and to high pH/alkalinity in CO3 /HCO3 -dominated exposures. Previous work with C. dubia also suggested a general ion toxicity involving all ions that was correlated with osmolarity. For fathead minnow, similar correlations were observed, but multiple mechanisms were indicated. At higher Ca, this general toxicity could be attributable to osmotic effects, but at lower Ca, osmolarity may be more a covariate than a cause, with this toxicity being related to a combined effect of ions other than via osmolarity. Environ Toxicol Chem 2022;41:2078-2094. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

A Multidimensional Matrix Model for Predicting the Effects of Male-Biased Sex Ratios on Fish Populations.

Laboratory experiments have established that exposure to certain endocrine-active substances prior to and/or during the period of sexual differentiation can lead to skewed sex ratios in fish. However, the potential long-term population impact of biased sex ratio depends on multiple factors including the life history of the species and whether the ratio is male or female-biased. In the present study, we describe a novel multidimensional, density-dependent matrix model that analyzes age class-structure of both males and females over time, allowing for the quantitative evaluation of the effects of biased sex ratio on population status. This approach can be used in conjunction with field monitoring efforts and/or laboratory testing to link effects on sex ratio due to chemical and/or nonchemical stressors to adverse outcomes in whole organisms and populations. For demonstration purposes, we applied the model to evaluate population trajectories for fathead minnow (Pimephales promelas) exposed to prochloraz, an aromatase inhibitor, during sexual differentiation. The model also was used to explore the population impact in a more realistic exposure scenario in which both adult and early life stages of fish are exposed concurrently to prochloraz, which, in addition to altering sex ratio during development, can decrease vitellogenin and egg production in adult females. For each exposure scenario, the model was used to analyze total population size, numbers of females and of males, and sex specific recruitment of the F1 generation. The present study illustrates the utility of multidimensional matrix population models for ecological risk assessment in terms of integrating effects across a population of an organism even when chemical effects on individuals are manifested via different pathways depending on life stage. Environ Toxicol Chem 2022;41:1066-1077. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

Characterizing toxicity pathways of fluoxetine to predict adverse outcomes in adult fathead minnows (Pimephales promelas).

Current ecotoxicity testing programs are impeded as they predominantly rely on slow and expensive animal tests measuring adverse outcomes. Therefore, new approach methodologies (NAMs) increasingly involve short-term mechanistic assays that employ molecular endpoints to predict adverse outcomes of regulatory relevance. This study aimed to elucidate the application of NAMs in adult fathead minnows using fluoxetine (FLX) as a model compound. Fish were exposed to three FLX concentrations (measured: 2.42, 10.7, and 56.7 µgL-1) and a control. After 96 h, molecular toxicity signatures were characterized using proteomics and transcriptomics analyses in livers and brains of a sub-set of fish. The remaining fish were sampled at 21 days and assessed for liver histopathology and morphometric measurements. Fecundity was monitored throughout the study. In the livers, 56.7 µgL-1 FLX caused enrichment of PPAR signaling in the proteome and fatty acid-related pathways in the transcriptome, potential upstream responses that led to lipid-type vacuolation of hepatocytes, observed via histopathology. Upregulated genes in the brain suggested alterations in serotonin-related signaling processes and reproductive behaviour, which may explain the observed significant decrease in fecundity. While the relationships between molecular responses and adverse outcomes remain complex, this research provided important insights into the mechanistic toxicity of FLX.

Effects of a 28-day early life stage exposure to carbaryl on fathead minnow long-term growth and reproduction.

The US Environmental Protection Agency conducts ecological risk assessments with a battery of fish toxicity tests that include acute, early life stage, and reproduction tests. While endpoints in these tests (survival, growth and reproduction) are conceptually related, because they are measured in separate exposures, the quantitative relationships between them are difficult to determine and largely ignored. In the current test, fathead minnows (FHM) were exposed for 28 days to 1 mg/L or 2 mg/L carbaryl, a well-studied carbamate insecticide, in early life stages and then reared in clean water until adulthood, when reproduction was assessed. Also. weekly growth measurements were taken throughout the test to determine growth rates during and after exposure. Growth curves derived from these measurements were then compared to the reproductive output. The data indicate that carbaryl reduced growth rate only for a brief time early in the exposure. However, this brief effect impacted overall growth into adulthood and lowered the reproductive output of exposed FHM. The effect of a transient exposure early in life to carbaryl could have later population-level impacts by causing mortality, lowering growth rates, and reducing reproductive output.

Exploratory analysis of the application of animal reduction approaches in proteomics: How much is enough?

Animal testing has long been the cornerstone of chemical safety assessments, but fish embryo assays represent an alter­native. Omics studies allow the examination of early molecular responses of organisms to environmental stressors, but reduction of animal use within this context has been overlooked. For proteomics, there is significant disparity and vari­ability in the organismal pool size used for studies, ranging from 1-1500 embryos per replicate for zebrafish alone. However, it is unknown if varying sample pool size results in differences in protein identifications. To examine whether the detected proteome changes depend on this variable, 3 pool sizes (5, 10 or 20 embryos or larvae per replicate) were compared using the two most common fish models with an appropriate biological replicate number determined by power analysis (n = 7). Data was acquired using MSe, resulting in 1,946 and 3,172 protein groups identified (1% false discovery rate) for fathead minnow and zebrafish, respectively. Proteins were not differentially expressed among pool sizes, and no significant difference was observed among the identified protein groups. However, for the fathead minnow, a decrease in the number of identified proteins was observed with increasing pool size, while a trend towards an increase in protein identifications was observed in zebrafish between the lowest and highest pool size. Taken together, our observations suggest that a proteome characterization experiment using these fish models can achieve comparable protein identifications using pool sizes of less than 5 organisms per replicate, assuming a protein requirement of 50 µg or less.

De Novo Assembly of the Nearly Complete Fathead Minnow Reference Genome Reveals a Repetitive but Compact Genome.

The fathead minnow is a widely used model organism in environmental toxicology. The lack of a high-quality fathead minnow reference genome, however, has severely hampered its uses in toxicogenomics. We present the de novo assembly and annotation of the fathead minnow genome using long PacBio reads, Bionano and Hi-C scaffolding data, and large RNA-sequencing data sets from different tissues and life stages. The new annotated fathead minnow reference genome has a scaffold N50 of 12.0 Mbp and a complete benchmarking universal single-copy orthologs score of 95.1%. The completeness of annotation for the new reference genome is comparable to that of the zebrafish GRCz11 reference genome. The fathead minnow genome, revealed to be highly repetitive and sharing extensive syntenic regions with the zebrafish genome, has a much more compact gene structure than the zebrafish genome. Particularly, comparative genomic analysis with zebrafish, mouse, and human showed that fathead minnow homologous genes are relatively conserved in exon regions but had strikingly shorter intron regions. The new fathead minnow reference genome and annotation data, publicly available from the National Center for Biotechnology Information and the University of California Santa Cruz genome browser, provides an essential resource for aquatic toxicogenomic studies in ecotoxicology and public health. Environ Toxicol Chem 2022;41:448-461. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

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.