Trait-based approach to bacterial growth efficiency.

Bacterial growth efficiency (BGE) is the proportion of assimilated carbon that is converted into biomass and reflects the balance between growth and energetic demands. Often measured as an aggregate property of the community, BGE is highly variable within and across ecosystems. To understand this variation, we first identified how species identity and resource type affect BGE using 20 bacterial isolates belonging to the phylum Proteobacteria that were enriched from north temperate lakes. Using a trait-based approach that incorporated genomic and phenotypic information, we characterized the metabolism of each isolate and tested for predicted trade-offs between growth rate and efficiency. A substantial amount of variation in BGE could be explained at broad (i.e., order, 20%) and fine (i.e., strain, 58%) taxonomic levels. While resource type was a relatively weak predictor across species, it explained >60% of the variation in BGE within a given species. A metabolic trade-off (between maximum growth rate and efficiency) and genomic features revealed that BGE may be a species-specific metabolic property. Our study suggests that genomic and phylogenetic information may help predict aggregate microbial community functions like BGE and the fate of carbon in ecosystems.

Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).

BACKGROUND: Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. OBJECTIVES: The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. METHODS: Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to 0.43-7.86µM PFOS, 7.87-120µM PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response (BMCT) modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to 7.86µM PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. RESULTS: Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, BMCT estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. DISCUSSION: This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667.

Monoassociation with bacterial isolates reveals the role of colonization, community complexity and abundance on locomotor behavior in larval zebrafish.

BACKGROUND: Across taxa, animals with depleted intestinal microbiomes show disrupted behavioral phenotypes. Axenic (i.e., microbe-free) mice, zebrafish, and fruit flies exhibit increased locomotor behavior, or hyperactivity. The mechanism through which bacteria interact with host cells to trigger normal neurobehavioral development in larval zebrafish is not well understood. Here, we monoassociated zebrafish with either one of six different zebrafish-associated bacteria, mixtures of these host-associates, or with an environmental bacterial isolate. RESULTS: As predicted, the axenic cohort was hyperactive. Monoassociation with three different host-associated bacterial species, as well as with the mixtures, resulted in control-like locomotor behavior. Monoassociation with one host-associate and the environmental isolate resulted in the hyperactive phenotype characteristic of axenic larvae, while monoassociation with two other host-associated bacteria partially blocked this phenotype. Furthermore, we found an inverse relationship between the total concentration of bacteria per larvae and locomotor behavior. Lastly, in the axenic and associated cohorts, but not in the larvae with complex communities, we detected unexpected bacteria, some of which may be present as facultative predators. CONCLUSIONS: These data support a growing body of evidence that individual species of bacteria can have different effects on host behavior, potentially related to their success at intestinal colonization. Specific to the zebrafish model, our results suggest that differences in the composition of microbes in fish facilities could affect the results of behavioral assays within pharmacological and toxicological studies.

Evaluation of Developmental Toxicity, Developmental Neurotoxicity, and Tissue Dose in Zebrafish Exposed to GenX and Other PFAS.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are a diverse class of industrial chemicals with widespread environmental occurrence. Exposure to long-chain PFAS is associated with developmental toxicity, prompting their replacement with short-chain and fluoroether compounds. There is growing public concern over the safety of replacement PFAS. OBJECTIVE: We aimed to group PFAS based on shared toxicity phenotypes. METHODS: Zebrafish were developmentally exposed to 4,8-dioxa-3H-perfluorononanoate (ADONA), perfluoro-2-propoxypropanoic acid (GenX Free Acid), perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluoro-n-octanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), or 0.4% dimethyl sulfoxide (DMSO) daily from 0-5 d post fertilization (dpf). At 6 dpf, developmental toxicity and developmental neurotoxicity assays were performed, and targeted analytical chemistry was used to measure media and tissue doses. To test whether aliphatic sulfonic acid PFAS cause the same toxicity phenotypes, perfluorobutanesulfonic acid (PFBS; 4-carbon), perfluoropentanesulfonic acid (PFPeS; 5-carbon), PFHxS (6-carbon), perfluoroheptanesulfonic acid (PFHpS; 7-carbon), and PFOS (8-carbon) were evaluated. RESULTS: PFHxS or PFOS exposure caused failed swim bladder inflation, abnormal ventroflexion of the tail, and hyperactivity at nonteratogenic concentrations. Exposure to PFHxA resulted in a unique hyperactivity signature. ADONA, PFESA1, or PFOA exposure resulted in detectable levels of parent compound in larval tissue but yielded negative toxicity results. GenX was unstable in DMSO, but stable and negative for toxicity when diluted in deionized water. Exposure to PFPeS, PFHxS, PFHpS, or PFOS resulted in a shared toxicity phenotype characterized by body axis and swim bladder defects and hyperactivity. CONCLUSIONS: All emerging fluoroether PFAS tested were negative for evaluated outcomes. Two unique toxicity signatures were identified arising from structurally dissimilar PFAS. Among sulfonic acid aliphatic PFAS, chemical potencies were correlated with increasing carbon chain length for developmental neurotoxicity, but not developmental toxicity. This study identified relationships between chemical structures and in vivo phenotypes that may arise from shared mechanisms of PFAS toxicity. These data suggest that developmental neurotoxicity is an important end point to consider for this class of widely occurring environmental chemicals. https://doi.org/10.1289/EHP5843.

Workflow for defining reference chemicals for assessing performance of in vitro assays.

Instilling confidence in use of in vitro assays for predictive toxicology requires evaluation of assay performance. Performance is typically assessed using reference chemicals--compounds with defined activity against the test system target. However, developing reference chemical lists has historically been very resource-intensive. We developed a semi-automated process for selecting and annotating reference chemicals across many targets in a standardized format and demonstrate the workflow here. A series of required fields defines the potential reference chemical: the in vitro molecular target, pathway, or phenotype affected; and the chemical's mode (e.g. agonist, antagonist, inhibitor). Activity information was computationally extracted into a database from multiple public sources including non-curated scientific literature and curated chemical-biological databases, resulting in the identification of chemical activity in 2995 biological targets. Sample data from literature sources covering 54 molecular targets ranging from data-poor to data-rich was manually checked for accuracy. Precision rates were 82.7% from curated data sources and 39.5% from automated literature extraction. We applied the final reference chemical lists to evaluating performance of EPA's ToxCast program in vitro bioassays. The level of support, i.e. the number of independent reports in the database linking a chemical to a target, was found to strongly correlate with likelihood of positive results in the ToxCast assays, although individual assay performance had considerable variation. This overall approach allows rapid development of candidate reference chemical lists for a wide variety of targets that can facilitate performance evaluation of in vitro assays as a critical step in imparting confidence in alternative approaches.