Impacts on Atlantic Killifish from Neurotoxicants: Genes, Behavior, and Population-Relevant Outcomes.

Molecular, cellular, and organismal alterations are important descriptors of toxic effects, but our ability to extrapolate and predict ecological risks is limited by the availability of studies that link measurable end points to adverse population relevant outcomes such as cohort survival and growth. In this study, we used laboratory gene expression and behavior data from two populations of Atlantic killifish Fundulus heteroclitus [one reference site (SCOKF) and one PCB-contaminated site (NBHKF)] to inform individual-based models simulating cohort growth and survival from embryonic exposures to environmentally relevant concentrations of neurotoxicants. Methylmercury exposed SCOKF exhibited brain gene expression changes in the si:ch211-186j3.6, si:dkey-21c1.4, scamp1, and klhl6 genes, which coincided with changes in feeding and swimming behaviors, but our models simulated no growth or survival effects of exposures. PCB126-exposed SCOKF had lower physical activity levels coinciding with a general upregulation in nucleic and cellular brain gene sets (BGS) and downregulation in signaling, nucleic, and cellular BGS. The NBHKF, known to be tolerant to PCBs, had altered swimming behaviors that coincided with 98% fewer altered BGS. Our models simulated PCB126 decreased growth in SCOKF and survival in SCOKF and NBHKF. Overall, our study provides a unique demonstration linking molecular and behavioral data to develop quantitative, testable predictions of ecological risk.

Altered Larval Yellow Perch Swimming Behavior Due to Methylmercury and PCB126 Detected Using Hidden Markov Chain Models.

Fish swimming behavior is a commonly measured response in aquatic ecotoxicology because behavior is considered a whole organism-level effect that integrates many sensory systems. Recent advancements in animal behavior models, such as hidden Markov chain models (HMM), suggest an improved analytical approach for toxicology. Using both new and traditional approaches, we examined the sublethal effects of PCB126 and methylmercury on yellow perch (YP) larvae (Perca flavescens) using three doses. Both approaches indicate larvae increase activity after exposure to either chemical. The middle methylmercury-dosed larvae showed multiple altered behavior patterns. First, larvae had a general increase in activity, typically performing more behavior states, more time swimming, and more swimming bouts per second. Second, when larvae were in a slow or medium swimming state, these larvae tended to switch between these states more often. Third, larvae swam slower during the swimming bouts. The upper PCB126-dosed larvae exhibited a higher proportion and a fast swimming state, but the total time spent swimming fast decreased. The middle PCB126-dosed larvae transitioned from fast to slow swimming states less often than the control larvae. These results indicate that developmental exposure to very low doses of these neurotoxicants alters YP larvae overall swimming behaviors, suggesting neurodevelopment alteration.

Triple-stage Quadrupole Mass Spectrometer to Determine Ubiquitously Present Per- and Polyfluorinated Alkyl Substances in Drinking Water at Part Per Trillion Levels Using Solid Phase Extraction Approach.

An accurate analytical method was developed to determine selected per- and polyfluorinated alkyl substances (PFAS) at the level of parts per trillion (ppt or ng/L) in drinking water. The method included a concentration step using solid phase extraction (SPE) approach in combination with a liquid chromatography-tandem mass spectrometry system (LC-MS/MS). This method was optimized and validated for the common PFAS contaminants in drinking water. An initial demonstration of capability was established with an acceptable initial calibration, minimum reporting limit (MRL), limit of detection (LOD), initial demonstration of low system background, and initial demonstration of precision (IDP). Isotopically labeled internal standards were used for quantification. Surrogate standards were used to monitor method performance. The current method will help in better understanding of PFAS crisis by providing an efficient measurement of PFAS in water. In this study, the recoveries of four surrogates were between 84 and 113%, and calculated limit of detection (DL) and minimum reporting limits (MRL) were generally 1.0-3.0 and 5-10 ng/L, respectively.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Sea lampreys elicit strong transcriptomic responses in the lake trout liver during parasitism.

BACKGROUND: The sea lamprey (Petromyzon marinus) is a jawless vertebrate that parasitizes fish as an adult and, with overfishing, was responsible for the decline in lake trout (Salvelinus namaycush) populations in the Great Lakes. While laboratory studies have looked at the rates of wounding on various fish hosts, there have been few investigations on the physiological effects of lamprey wounding on the host. In the current study, two morphotypes of lake trout, leans and siscowets, were parasitized in the laboratory by sea lampreys and the liver transcriptomes of parasitized and nonparasitized fish were analyzed by RNA-seq (DESeq2 and edgeR) to determine which genes and gene pathways (Ingenuity Pathway Analysis) were altered by lamprey parasitism. RESULTS: Overall, genes encoding molecules involved in catalytic (e.g., enzymatic) and binding activities (factors and regulators) predominated the regulated gene lists. In siscowets, the top upregulated gene was growth arrest and DNA-damage-inducible protein and for leans it was interleukin-18-binding protein. In leans, the most significantly downregulated gene was UDP-glucuronosyltransferase 2A2 - DESeq2 or phosphotriesterase related - edgeR. For siscowets, the top downregulated gene was C-C motif chemokine 19 - DESeq2 or GTP-binding protein Rhes - edgeR. Gene pathways associated with inflammatory-related responses or factors (cytokines, chemokines, oxidative stress, apoptosis) were regulated following parasitism in both morphotypes. However, pathways related to energy metabolism (glycolysis, gluconeogenesis, lipolysis, lipogenesis) were also regulated. These pathways or the intensity or direction (up/downregulation) of regulation were different between leans and siscowets. Finally, one of the most significantly downregulated pathways in both leans and siscowets was the kynurenine (tryptophan degradation) pathway. CONCLUSIONS: The results indicate a strong transcriptional response in the lake trout to lamprey parasitism that entails genes involved in the regulation of inflammation and cellular damage. Responses to energy utilization as well as hydromineral balance also occurred indicating an adjustment in the host to energy demands and osmotic imbalances during parasitism. Given the role of the kynurenine pathway in promoting immunotolerance in mammals, the downregulation observed in this pathway during parasitism may signify an attempt by the host to inhibit any feedback suppression of the immune response to the lamprey.

Parental Whole Life Cycle Exposure to Dietary Methylmercury in Zebrafish (Danio rerio) Affects the Behavior of Offspring.

Methylmercury (MeHg) is an established neurotoxicant of concern to fish-eating organisms. While most studies have focused on the fish consumers, much less is known about the effects of MeHg on the fish themselves, especially following exposures to chronic and environmentally relevant scenarios. Here we evaluated the behavioral effects of developmental MeHg insult by exposing parental generations of zebrafish to an environmentally realistic MeHg dietary concentration (1 ppm) and two higher concentrations (3 and 10 ppm) throughout their whole life span. Upon reaching adulthood, their offspring were analyzed through a series of behavioral tests, including the visual-motor response (VMR) assay, analysis of spontaneous swimming and evaluation of foraging efficiency. The VMR assay identified decreased locomotor output in the 6 day postfertilization (dpf) offspring of fish exposed to 3 and 10 ppm MeHg. However, in a second test 7 dpf fish revealed an increase in locomotor activity in all MeHg exposures tested. Increases in locomotion continued to be observed until 16 dpf, which coincided with increased foraging efficiency. These results suggest an association between MeHg and hyperactivity, and imply that fish chronically exposed to MeHg in the wild may be vulnerable to predation.

Relating fish health and reproductive metrics to contaminant bioaccumulation at the Tennessee Valley Authority Kingston coal ash spill site.

A 4.1 million m(3) coal ash release into the Emory and Clinch rivers in December 2008 at the Tennessee Valley Authority's Kingston Fossil Plant in east Tennessee, USA, prompted a long-term, large-scale biological monitoring effort to determine if there are chronic effects of this spill on resident biota. Because of the magnitude of the ash spill and the potential for exposure to coal ash-associated contaminants [e.g., selenium (Se), arsenic (As), and mercury (Hg)] which are bioaccumulative and may present human and ecological risks, an integrative, bioindicator approach was used. Three species of fish were monitored-bluegill (Lepomis macrochirus), redear sunfish (L. microlophus), and largemouth bass (Micropterus salmoides)-at ash-affected and reference sites annually for 5 years following the spill. On the same individual fish, contaminant burdens were measured in various tissues, blood chemistry parameters as metrics of fish health, and various condition and reproduction indices. A multivariate statistical approach was then used to evaluate relationships between contaminant bioaccumulation and fish metrics to assess the chronic, sub-lethal effects of exposure to the complex mixture of coal ash-associated contaminants at and around the ash spill site. This study suggests that while fish tissue concentrations of some ash-associated contaminants are elevated at the spill site, there was no consistent evidence of compromised fish health linked with the spill. Further, although relationships between elevated fillet burdens of ash-associated contaminants and some fish metrics were found, these relationships were not indicative of exposure to coal ash or spill sites. The present study adds to the weight of evidence from prior studies suggesting that fish populations have not incurred significant biological effects from spilled ash at this site: findings that are relevant to the current national discussions on the safe disposal of coal ash waste.

Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011.

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May-July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.

Factors affecting recruitment into psychiatry: a canadian experience.

OBJECTIVE: There is a projected shortage of psychiatrists in Canada in forthcoming years. This study assessed factors in medical school education that are associated with students selecting psychiatry first and matching as a discipline. METHOD: The Canadian Organization of Undergraduate Psychiatry Educators (COUPE) conducted telephone interviews and sent e-mail questionnaires to the 17 medical schools across Canada; all schools provided data for 2012. Relevant data were obtained from the Canadian Resident Matching Service. Statistics were performed using v12 STATA program, and significance was set at a p value of <0.05. RESULTS: Medical student enrollment ranged from 54 to 266 students (mean = 158 ± 16). Of these students, 4.9 ± 0.6 % ranked psychiatry as their first choice for residency. Final match results yielded similar numbers at 5.0 ± 0.6 %. Ten out of 17 programs filled all psychiatry residency positions, whereas the remaining 7 programs had vacancy rates from 5 to 100 % (mean = 43.4 ± 15.1 %). Medical students were exposed to an average of 2.8 ± 0.5 pre-clerkship psychiatry weeks and 6.2 ± 0.3 clerkship weeks. Linear regression analysis demonstrated that the percentage of graduating medical students entering a psychiatry residency program could be predicted from the number of weeks of pre-clerkship exposure (p = 0.01; R(2) = 0.36) but not from the number of clerkship weeks (p = 0.74). CONCLUSIONS: This study indicates that the duration of pre-clerkship exposure to psychiatry predicts the number of students selecting psychiatry as their first choice as a discipline. Thus, increasing the duration of pre-clerkship exposure may increase the enrollment of medical students into psychiatry.

Identification of 50 class D ß-lactamases and 65 Acinetobacter-derived cephalosporinases in Acinetobacter spp.

Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D ß-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D ß-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D ß-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of ß-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of ß-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

Remediation practices in Canadian psychiatry clerkships.

OBJECTIVES: There is a lack of information regarding interventions for undergraduate students at Canadian medical schools who require remediation during their psychiatric training. The need for a theoretical framework to guide remediation has been identified. In this study, we sought to characterize remediation taking place in undergraduate psychiatry education, particularly during clerkship. A secondary goal was to ascertain whether those responsible for remediation were aware of the stages of change (transtheoretical) model, and whether they formally incorporated this framework (or another) into their remediation processes. METHODS: A short six-question survey on remediation practices was e-mailed to educators responsible for undergraduate psychiatry education at all 17 Canadian medical schools as identified through the Canadian Organization of Undergraduate Psychiatry Educators (COUPE). RESULTS: The response rate was 67 %. Respondents' overall impressions of their school's remediation process were that it was "highly effective" (25 %), or "somewhat effective" (67 %); 8 % reported being unsure of its effectiveness. While 75 % of survey respondents were aware of the stages of change model, only 17 % reported using this framework: no alternate theoretical frameworks were reported. CONCLUSIONS: The most common form of evaluation was multiple-choice question (MCQ) exams, and the most common form of remediation was exam rewrites. There is little information regarding the long-term outcomes of remediation, and further research would be useful in formulating recommendations regarding best practices.

How Model Organisms and Model Uncertainty Impact Our Understanding of the Risk of Sublethal Impacts of Toxicants to Survival and Growth of Ecologically Relevant Species.

Understanding how sublethal impacts of toxicants affect population-relevant outcomes for organisms is challenging. We tested the hypotheses that the well-known sublethal impacts of methylmercury (MeHg) and a polychlorinated biphenyl (PCB126) would have meaningful impacts on cohort growth and survival in yellow perch (Perca flavescens) and Atlantic killifish (Fundulus heteroclitus) populations, that inclusion of model uncertainty is important for understanding the sublethal impacts of toxicants, and that a model organism (zebrafish Danio rerio) is an appropriate substitute for ecologically relevant species (yellow perch, killifish). Our simulations showed that MeHg did not have meaningful impacts on growth or survival in a simulated environment except to increase survival and growth in low mercury exposures in yellow perch and killifish. For PCB126, the high level of exposure resulted in lower survival for killifish only. Uncertainty analyses increased the variability and lowered average survival estimates across all species and toxicants, providing a more conservative estimate of risk. We demonstrate that using a model organism instead of the species of interest does not necessarily give the same results, suggesting that using zebrafish as a surrogate for yellow perch and killifish may not be appropriate for predicting contaminant impacts on larval cohort growth and survival in ecologically relevant species. Our analysis also reinforces the notion that uncertainty analyses are necessary in any modeling assessment of the impacts of toxicants on a population because it provides a more conservative, and arguably realistic, estimate of impact. Environ Toxicol Chem 2024;43:2122-2133. © 2024 SETAC.

MicroRNAs induced in melanoma treated with combination targeted therapy of Temsirolimus and Bevacizumab.

BACKGROUND: Targeted therapies directed at commonly overexpressed pathways in melanoma have clinical activity in numerous trials. Little is known about how these therapies influence microRNA (miRNA) expression, particularly with combination regimens. Knowledge of miRNAs altered with treatment may contribute to understanding mechanisms of therapeutic effects, as well as mechanisms of tumor escape from therapy. We analyzed miRNA expression in metastatic melanoma tissue samples treated with a novel combination regimen of Temsirolimus and Bevacizumab. Given the preliminary clinical activity observed with this combination regimen, we hypothesized that we would see significant changes in miRNA expression with combination treatment. METHODS: Using microarray analysis we analyzed miRNA expression levels in melanoma samples from a Cancer Therapy Evaluation Program-sponsored phase II trial of combination Temsirolimus and Bevacizumab in advanced melanoma, which elicited clinical benefit in a subset of patients. Pre-treatment and post-treatment miRNA levels were compared using paired t-tests between sample groups (patients), using a p-value < 0.01 for significance. RESULTS: microRNA expression remained unchanged with Temsirolimus alone; however, expression of 15 microRNAs was significantly upregulated (1.4 to 2.5-fold) with combination treatment, compared to pre-treatment levels. Interestingly, twelve of these fifteen miRNAs possess tumor suppressor capabilities. We identified 15 putative oncogenes as potential targets of the 12 tumor suppressor miRNAs, based on published experimental evidence. For 15 of 25 miRNA-target mRNA pairings, changes in gene expression from pre-treatment to post-combination treatment samples were inversely correlated with changes in miRNA expression, supporting a functional effect of those miRNA changes. Clustering analyses based on selected miRNAs suggest preliminary signatures characteristic of clinical response to combination treatment and of tumor BRAF mutational status. CONCLUSIONS: To our knowledge, this is the first study analyzing miRNA expression in pre-treatment and post-treatment human metastatic melanoma tissue samples. This preliminary investigation suggests miRNAs that may be involved in the mechanism of action of combination Temsirolimus and Bevacizumab in metastatic melanoma, possibly through inhibition of oncogenic pathways, and provides the preliminary basis for further functional studies of these miRNAs.

The consequences of sea lamprey parasitism on lake trout energy budgets.

Parasitism is an energetically costly event for host species. Dynamic energy budget (DEB) theory describes the metabolic dynamics of an individual organism through its lifetime. Models derived from DEB theory specify how an organism converts food to reserves (maintenance-free energy available for metabolism) and allocates mobilized reserves to maintenance, growth (increase of structural body mass) and maturation or reproduction. DEB models thus provide a useful approach to describe the consequences of parasitism for host species. We developed a DEB model for siscowet lake trout and modeled the impact of sea lamprey parasitism on growth and reproduction using data collected from studies documenting the long-term effects following a non-lethal sea lamprey attack. The model was parameterized to reflect the changes in allocation of energy towards growth and reproduction observed in lake trout following sea lamprey parasitism and includes an estradiol module that describes the conversion of reproductive reserves to ovarian mass based on estradiol concentration. In our DEB model, parasitism increased somatic and maturity maintenance costs, reduced estradiol and decreased the estradiol-mediated conversion efficiency of reproductive reserves to ovarian mass. Muscle lipid composition of lake trout influenced energy mobilization from the reserve (efficiency of converting reserves allocated to reproduction into eggs) and reproductive efficiency. These model changes accurately reflect observed empirical changes to ovarian mass and growth. This model provides a plausible explanation of the energetic mechanisms that lead to skipped spawning following sea lamprey parasitism and could be used in population models to explore sublethal impacts of sea lamprey parasitism and other stressors on population dynamics.

The Influence of Life History on the Response to Parasitism: Differential Response to Non-Lethal Sea Lamprey Parasitism by Two Lake Charr Ecomorphs.

The energetic demands of stressors like parasitism require hosts to reallocate energy away from normal physiological processes to survive. Life history theory provides predictions about how hosts will reallocate energy following parasitism, but few studies provide empirical evidence to test these predictions. We examined the sub-lethal effects of sea lamprey parasitism on lean and siscowet lake charr, two ecomorphs with different life history strategies. Leans are shorter lived, faster growing, and reach reproductive maturity earlier than siscowets. Following a parasitism event of 4 days, we assessed changes to energy allocation by monitoring endpoints related to reproduction, energy storage, and growth. Results indicate that lean and siscowet lake charr differ considerably in their response to parasitism. Severely parasitized leans slightly increased their reproductive effort and maintained growth and energy storage, consistent with expectations based on life history that leans are less likely to survive parasitism and have shorter lifespans than siscowets making investing in immediate reproduction more adaptive. Siscowets nearly ceased reproduction following severe parasitism and showed evidence of altered energy storage, consistent with a strategy that favors maximizing long-term reproductive success. These findings suggest that life history can be used to generalize stressor response between populations and can aid management efforts.

Biological Networks across Scales-The Theoretical and Empirical Foundations for Time-Varying Complex Networks that Connect Structure and Function across Levels of Biological Organization.

Many biological systems across scales of size and complexity exhibit a time-varying complex network structure that emerges and self-organizes as a result of interactions with the environment. Network interactions optimize some intrinsic cost functions that are unknown and involve for example energy efficiency, robustness, resilience, and frailty. A wide range of networks exist in biology, from gene regulatory networks important for organismal development, protein interaction networks that govern physiology and metabolism, and neural networks that store and convey information to networks of microbes that form microbiomes within hosts, animal contact networks that underlie social systems, and networks of populations on the landscape connected by migration. Increasing availability of extensive (big) data is amplifying our ability to quantify biological networks. Similarly, theoretical methods that describe network structure and dynamics are being developed. Beyond static networks representing snapshots of biological systems, collections of longitudinal data series can help either at defining and characterizing network dynamics over time or analyzing the dynamics constrained to networked architectures. Moreover, due to interactions with the environment and other biological systems, a biological network may not be fully observable. Also, subnetworks may emerge and disappear as a result of the need for the biological system to cope with for example invaders or new information flows. The confluence of these developments renders tractable the question of how the structure of biological networks predicts and controls network dynamics. In particular, there may be structural features that result in homeostatic networks with specific higher-order statistics (e.g., multifractal spectrum), which maintain stability over time through robustness and/or resilience to perturbation. Alternative, plastic networks may respond to perturbation by (adaptive to catastrophic) shifts in structure. Here, we explore the opportunity for discovering universal laws connecting the structure of biological networks with their function, positioning them on the spectrum of time-evolving network structure, that is, dynamics of networks, from highly stable to exquisitely sensitive to perturbation. If such general laws exist, they could transform our ability to predict the response of biological systems to perturbations-an increasingly urgent priority in the face of anthropogenic changes to the environment that affect life across the gamut of organizational scales.

Key challenges and developments in wildlife ecological risk assessment: Problem formulation.

Problem formulation (PF) is a critical initial step in planning risk assessments for chemical exposures to wildlife, used either explicitly or implicitly in various jurisdictions to include registration of new pesticides, evaluation of new and existing chemicals released to the environment, and characterization of impact when chemical releases have occurred. Despite improvements in our understanding of the environment, ecology, and biological sciences, few risk assessments have used this information to enhance their value and predictive capabilities. In addition to advances in organism-level mechanisms and methods, there have been substantive developments that focus on population- and systems-level processes. Although most of the advances have been recognized as being state-of-the-science for two decades or more, there is scant evidence that they have been incorporated into wildlife risk assessment or risk assessment in general. In this article, we identify opportunities to consider elevating the relevance of wildlife risk assessments by focusing on elements of the PF stage of risk assessment, especially in the construction of conceptual models and selection of assessment endpoints that target population- and system-level endpoints. Doing so will remain consistent with four established steps of existing guidance: (1) establish clear protection goals early in the process; (2) consider how data collection using new methods will affect decisions, given all possibilities, and develop a decision plan a priori; (3) engage all relevant stakeholders in creating a robust, holistic conceptual model that incorporates plausible stressors that could affect the targets defined in the protection goals; and (4) embrace the need for iteration throughout the PF steps (recognizing that multiple passes may be required before agreeing on a feasible plan for the rest of the risk assessment). Integr Environ Assess Manag 2022;00:1-16. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.