Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Implementation of Zebrafish Ontologies for Toxicology Screening.

Toxicological evaluation of chemicals using early-life stage zebrafish (Danio rerio) involves the observation and recording of altered phenotypes. Substantial variability has been observed among researchers in phenotypes reported from similar studies, as well as a lack of consistent data annotation, indicating a need for both terminological and data harmonization. When examined from a data science perspective, many of these apparent differences can be parsed into the same or similar endpoints whose measurements differ only in time, methodology, or nomenclature. Ontological knowledge structures can be leveraged to integrate diverse data sets across terminologies, scales, and modalities. Building on this premise, the National Toxicology Program's Systematic Evaluation of the Application of Zebrafish in Toxicology undertook a collaborative exercise to evaluate how the application of standardized phenotype terminology improved data consistency. To accomplish this, zebrafish researchers were asked to assess images of zebrafish larvae for morphological malformations in two surveys. In the first survey, researchers were asked to annotate observed malformations using their own terminology. In the second survey, researchers were asked to annotate the images from a list of terms and definitions from the Zebrafish Phenotype Ontology. Analysis of the results suggested that the use of ontology terms increased consistency and decreased ambiguity, but a larger study is needed to confirm. We conclude that utilizing a common data standard will not only reduce the heterogeneity of reported terms but increases agreement and repeatability between different laboratories. Thus, we advocate for the development of a zebrafish phenotype atlas to help laboratories create interoperable, computable data.

Perfluorooctanesulfonic Acid-Induced Toxicity on Zebrafish Embryos in the Presence or Absence of the Chorion.

Perfluorooctanesulfonic acid (PFOS) is a perfluorinated compound used in many industrial and consumer products. It has been linked to a broad range of adverse effects in several species, including zebrafish (Danio rerio). The zebrafish embryo is a widely used vertebrate model to elucidate potential adverse effects of chemicals because it is amenable to medium and high throughput. However, there is limited research on the full extent of the impact the chorion has on those effects. Results from the present study indicate that the presence of the chorion affected the timing and incidence of mortality as well as morphometric endpoints such as spinal curvature and swim bladder inflation in zebrafish embryos exposed to PFOS. Furthermore, removal of the chorion prior to exposure resulted in a lower threshold of sensitivity to PFOS for effects on transcriptional expression within the peroxisome proliferator-activated receptor (PPAR) nuclear signaling pathway. Perturbation of PPAR pathway gene expression can result in disruption of metabolic signaling and regulation, which can adversely affect development, energy availability, and survival. It can be concluded that removal of the chorion has significant effects on the timing and incidence of impacts associated with PFOS exposure, and more research is warranted to fully elucidate the protective role of the chorion and the critical timing of these events. Environ Toxicol Chem 2021;40:780-791. Published 2020. This article is a US Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Fungal and herbivore elicitation of the novel maize sesquiterpenoid, zealexin A4, is attenuated by elevated CO2.

MAIN CONCLUSION: Chemical isolation and NMR-based structure elucidation revealed a novel keto-acidic sesquiterpenoid, termed zealexin A4 (ZA4). ZA4 is elicited by pathogens and herbivory, but attenuated by heightened levels of CO 2 . The identification of the labdane-related diterpenoids, termed kauralexins and acidic sesquiterpenoids, termed zealexins, demonstrated the existence of at least ten novel stress-inducible maize metabolites with diverse antimicrobial activity. Despite these advances, the identity of co-occurring and predictably related analytes remains largely unexplored. In the current effort, we identify and characterize the first sesquiterpene keto acid derivative of ß-macrocarpene, named zealexin A4 (ZA4). Evaluation of diverse maize inbreds revealed that ZA4 is commonly produced in maize scutella during the first 14 days of seedling development; however, ZA4 production in the scutella was markedly reduced in seedlings grown in sterile soil. Elevated ZA4 production was observed in response to inoculation with adventitious fungal pathogens, such as Aspergillus flavus and Rhizopus microsporus, and a positive relationship between ZA4 production and expression of the predicted zealexin biosynthetic genes, terpene synthases 6 and 11 (Tps6 and Tps11), was observed. ZA4 exhibited significant antimicrobial activity against the mycotoxigenic pathogen A. flavus; however, ZA4 activity against R. microsporus was minimal, suggesting the potential of some fungi to detoxify ZA4. Significant induction of ZA4 production was also observed in response to infestation with the stem tunneling herbivore Ostrinia nubilalis. Examination of the interactive effects of elevated CO2 (E-CO2) on both fungal and herbivore-elicited ZA4 production revealed significantly reduced levels of inducible ZA4 accumulation, consistent with a negative role for E-CO2 on ZA4 production. Collectively, these results describe a novel ß-macrocarpene-derived antifungal defense in maize and expand the established diversity of zealexins that are differentially regulated in response to biotic/abiotic stress.

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR.

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard(®) (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.

Characterization of the Maize Chitinase Genes and Their Effect on Aspergillus flavus and Aflatoxin Accumulation Resistance.

Maize (Zea mays L.) is a crop of global importance, but prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need for intervention by farmers. Chitinolytic enzymes respond to attack by potential pathogens and have been demonstrated to increase insect and fungal resistance in plants. Here, all chitinase genes in the maize genome were characterized via sequence diversity and expression patterns. Recent evolution within this gene family was noted. Markers from within each gene were developed and used to map the phenotypic effect on resistance of each gene in up to four QTL mapping populations and one association panel. Seven chitinase genes were identified that had alleles associated with increased resistance to aflatoxin accumulation and A. flavus infection in field grown maize. The chitinase in bin 1.05 identified a new and highly significant QTL, while chitinase genes in bins 2.04 and 5.03 fell directly beneath the peaks of previously published QTL. The expression patterns of these genes corroborate possible grain resistance mechanisms. Markers from within the gene sequences or very closely linked to them are presented to aid in the use of marker assisted selection to improve this trait.

Relating significance and relations of differentially expressed genes in response to Aspergillus flavus infection in maize.

Aspergillus flavus is a pathogenic fungus infecting maize and producing aflatoxins that are health hazards to humans and animals. Characterizing host defense mechanism and prioritizing candidate resistance genes are important to the development of resistant maize germplasm. We investigated methods amenable for the analysis of the significance and relations among maize candidate genes based on the empirical gene expression data obtained by RT-qPCR technique from maize inbred lines. We optimized a pipeline of analysis tools chosen from various programs to provide rigorous statistical analysis and state of the art data visualization. A network-based method was also explored to construct the empirical gene expression relational structures. Maize genes at the centers in the network were considered as important candidate genes for maize DNA marker studies. The methods in this research can be used to analyze large RT-qPCR datasets and establish complex empirical gene relational structures across multiple experimental conditions.

Identification of maize genes associated with host plant resistance or susceptibility to Aspergillus flavus infection and aflatoxin accumulation.

BACKGROUND: Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation. RESULTS: Genome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E. CONCLUSION: Maize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.

A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize.

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.