CeO2 nanoparticle dose and exposure modulate soybean development and plant-mediated responses in root-associated bacterial communities.

Agricultural soils are increasingly undergoing inadvertent and purposeful exposures to engineered CeO2 nanoparticles (NPs), which can impact crops and root-associated microbial communities. However, interactions between NP concentration and exposure duration on plant-mediated responses of root-associated bacterial communities are not well understood. Soybeans seedlings were grown in soil with uncoated NPs added at concentrations of 0, 1 or 100 mg kg-1. Total soil exposure durations were either 190 days, starting 106 days before planting or 84 days with NP amendments coinciding with planting. We assessed plant development, bacterial diversity, differential abundance and inferred functional changes across rhizosphere, rhizoplane, and root tissue compartments. Plant non-monotonic dose responses were mirrored in bacterial communities. Most notably, effects were magnified in the rhizoplane under low-dose, short-exposures. Enriched metabolic pathways were primarily related to biosynthesis and degradation/utilization/assimilation, rather than responses to metals or oxidative stress. Our results indicate that plant-mediated bacterial responses were greater than direct NP impacts. Also, we identify needs for modeling non-monotonic legume stress responses that account for coinfection with mutualistic and parasitic bacteroids. Our findings provide new insights regarding effects of applications of soil amendments such as biosolids containing NPs or nano-enabled formulations used in cultivation of legumes and other crops.

Using eRNA/eDNA metabarcoding to detect community-level impacts of nanoplastic exposure to benthic estuarine ecosystems.

Plastic particles are ubiquitous in marine systems and fragment into smaller pieces, such as nanoplastics (NPs). The effects of NPs on marine organisms are of growing concern but are not well understood. Marine sediments act as a sink for many contaminants, like microplastics, and are rich habitats for benthic micro- and meiofauna which are ecologically-important components of marine food webs; however, little is known about the sensitivities of specific organisms to NPs or the effects on community diversity and composition. Utilizing molecular methods, such as metabarcoding of environmental DNA/RNA, allows for the rapid and comprehensive detection of microscopic organisms via high-throughput sequencing to assess adverse effects at the community level. The objective of this study was to use a metabarcoding approach to investigate the effects of NPs on benthic micro- and meiofaunal community diversity. Mesocosms were created with sediment cores collected from the Narrow River estuary (Rhode Island, USA) and exposed to 900 nm diameter weathered polystyrene beads at concentrations of 0.1, 1, 10, or 100 mg/kg dry weight in sediment for two weeks. Following exposure, RNA and DNA were co-extracted from the sediment, RNA was reverse-transcribed, 18S and COI markers were PCR-amplified, and amplicons were sequenced on an Illumina MiSeq. Using the 18S marker and eRNA template, increases to α-diversity and significant differences to ß-diversity were observed in the highest NP exposures relative to the control. Observed differences in community composition were driven by the differential abundance of several types of protists and arthropods. Significant dose-dependent shifts in composition were observed in ß-diversity Jaccard and Unweighted-Unifrac metrics with the 18S marker using the RNA template. To our knowledge, this is the first demonstration of a dose-response relationship for NPs at a community level, and it highlights the value of using community-level endpoints to assess environmental impacts of nanoparticles.

Environmental RNA as a Tool for Marine Community Biodiversity Assessments.

Microscopic organisms are often overlooked in traditional diversity assessments due to the difficulty of identifying them based on morphology. Metabarcoding is a method for rapidly identifying organisms where Environmental DNA (eDNA) is used as a template. However, legacy DNA is problematically detected from organisms no longer in the environment during sampling. Environmental RNA (eRNA), which is only produced by living organisms, can also be collected from environmental samples and used for metabarcoding. The aim of this study was to determine differences in community composition and diversity between eRNA and eDNA templates for metabarcoding. Using mesocosms containing field-collected communities from an estuary, RNA and DNA were co-extracted from sediment, libraries were prepared for two loci (18S and COI), and sequenced using an Illumina MiSeq. Results show a higher number of unique sequences detected from eRNA in both markers and higher α-diversity compared to eDNA. Significant differences between eRNA and eDNA for all ß-diversity metrics were also detected. This study is the first to demonstrate community differences detected with eRNA compared to eDNA from an estuarine system and illustrates the broad applications of eRNA as a tool for assessing benthic community diversity, particularly for environmental conservation and management applications.

Focused Microbiome Shifts in Reconstructed Wetlands Correlated with Elevated Copper Concentrations Originating from Micronized Copper Azole-Treated Wood.

Micronized copper (Cu) azole (MCA) wood preservative formulations include Cu in nano form, and relatively little is known about longer term effects of Cu leached from MCA into wetland ecosystems. We tested the hypothesis that changes in soil microbiomes within reconstructed freshwater wetlands will be associated with exposure to elevated Cu concentrations originating from immersed MCA-treated wood stakes. Eight replicate communities were assembled with Willamette Valley (OR, USA) flood plain soil and clonally propagated wetland plants within mesocosms. Inundated communities were equilibrated for 5 months before installation of MCA or control southern yellow pine stakes (n = 4 communities/experimental group). Soil samples were collected for 16S and internal transcribed spacer amplicon sequencing to quantify responses in prokaryotes and eukaryotes, respectively, at 15 time points, spanning two simulated seasonal dry downs, for up to 678 days. Physiochemical properties of water and soil were monitored at 20 and 12 time points respectively, over the same period. For both taxonomic groups of organisms, phylogenetic diversity increased and was positively correlated with elapsed days. Furthermore, there was significant divergence among eukaryotes during the second year based on experimental group. Although the composition of taxa underwent succession over time, there was significantly reduced relative abundance of sequence variants from Gomphonema diatoms and Scutellinia fungi in communities where MCA wood stakes were present compared with the controls. These focused microbiome shifts were positively correlated with surface water Cu and soil Cu concentrations, which were significantly elevated in treated communities. The reconstructed communities were effective systems for assessing potential impacts to wetland microbiomes after exposure to released copper. The results further inform postcommercialization risk assessments on MCA-treated wood. Environ Toxicol Chem 2021;40:3351-3368. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

Large-scale implementation of standardized quantitative real-time PCR fecal source identification procedures in the Tillamook Bay Watershed.

Fecal pollution management remains one of the biggest challenges for water quality authorities worldwide. Advanced fecal pollution source identification technologies are now available that can provide quantitative information from many animal groups. As public interest in these methodologies grows, it is vital to use standardized procedures with clearly defined data acceptance metrics and conduct field studies demonstrating the use of these techniques to help resolve real-world water quality challenges. Here we apply recently standardized human-associated qPCR methods with custom data acceptance metrics (HF183/BacR287 and HumM2), along with established procedures for ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution sources to (i) demonstrate the feasibility of implementing standardized qPCR procedures in a large-scale field study, and (ii) characterize trends in fecal pollution sources in the research area. A total of 602 water samples were collected over a one-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries in the Tillamook Bay Watershed (OR, USA). Host-associated qPCR results were combined with high-resolution geographic information system (GIS) land use and general indicator bacteria (E. coli) measurements to elucidate water quality fecal pollution trends. Results demonstrate the feasibility of implementing standardized fecal source identification qPCR methods with established data acceptance metrics in a large-scale field study leading to new investigative leads suggesting that elevated E. coli levels may be linked to specific pollution sources and land use activities in the Tillamook Bay Watershed.

Douglas-Fir ( Pseudotsuga menziesii (Mirb.) Franco) Transcriptome Profile Changes Induced by Diesel Emissions Generated with CeO2 Nanoparticle Fuel Borne Catalyst.

It is important to understand molecular effects on plants exposed to compounds released from use of products containing engineered nanomaterials. Here, we present mRNA sequencing data on transcriptome impacts to Douglas-fir following 2 weeks of sublethal exposure to 30:1 diluted airborne emissions released from combustion of diesel fuel containing engineered CeO2 nanoparticle catalysts (DECe). Our hypothesis was that chamber exposure to DECe would induce distinct transcriptome changes in seedling needles compared with responses to conventional diesel exhaust (DE) or filtered DECe Gas Phase. Significantly increased uptake/binding of Ce in needles of DECe treated seedlings was 2.7X above background levels and was associated with altered gene expression patterns. All 225 Blast2GO gene ontologies (GOs) enriched by up-regulated DECe transcripts were nested within GOs for DE, however, 29 of 31 enriched GOs for down-regulated DECe transcripts were unique. MapMan analysis also identified three pathways enriched with DECe down-regulated transcripts. There was prominent representation of genes with attenuated expression in transferase, transporter, RNA regulation and protein degradation GOs and pathways. CeO2 nanoparticle additive decreased and shifted molecular impact of diesel emissions. Wide-spread use of such products and chronic environmental exposure to DECe may adversely affect plant physiology and development.

A comprehensive framework for evaluating the environmental health and safety implications of engineered nanomaterials.

Engineered nanomaterials (ENM) are a growing aspect of the global economy, and their safe and sustainable development, use, and eventual disposal requires the capability to forecast and avoid potential problems. This review provides a framework to evaluate the health and safety implications of ENM releases into the environment, including purposeful releases such as for antimicrobial sprays or nano-enabled pesticides, and inadvertent releases as a consequence of other intended applications. Considerations encompass product life cycles, environmental media, exposed populations, and possible adverse outcomes. This framework is presented as a series of compartmental flow diagrams that serve as a basis to help derive future quantitative predictive models, guide research, and support development of tools for making risk-based decisions. After use, ENM are not expected to remain in their original form due to reactivity and/or propensity for hetero-agglomeration in environmental media. Therefore, emphasis is placed on characterizing ENM as they occur in environmental or biological matrices. In addition, predicting the activity of ENM in the environment is difficult due to the multiple dynamic interactions between the physical/chemical aspects of ENM and similarly complex environmental conditions. Others have proposed the use of simple predictive functional assays as an intermediate step to address the challenge of using physical/chemical properties to predict environmental fate and behavior of ENM. The nodes and interactions of the framework presented here reflect phase transitions that could be targets for development of such assays to estimate kinetic reaction rates and simplify model predictions. Application, refinement, and demonstration of this framework, along with an associated knowledgebase that includes targeted functional assay data, will allow better de novo predictions of potential exposures and adverse outcomes.

Molecular and physiological responses to titanium dioxide and cerium oxide nanoparticles in Arabidopsis.

Changes in tissue transcriptomes and productivity of Arabidopsis thaliana were investigated during exposure of plants to 2 widely used engineered metal oxide nanoparticles, titanium dioxide (nano-titania) and cerium dioxide (nano-ceria). Microarray analyses confirmed that exposure to either nanoparticle altered the transcriptomes of rosette leaves and roots, with comparatively larger numbers of differentially expressed genes found under nano-titania exposure. Nano-titania induced more differentially expressed genes in rosette leaves, whereas roots had more differentially expressed genes under nano-ceria exposure. MapMan analyses indicated that although nano-titania up-regulated overall metabolism in both tissues, metabolic processes under nano-ceria remained mostly unchanged. Gene enrichment analysis indicated that both nanoparticles mainly enriched ontology groups such as responses to stress (abiotic and biotic), and defense responses (pathogens), and responses to endogenous stimuli (hormones). Nano-titania specifically induced genes associated with photosynthesis, whereas nano-ceria induced expression of genes related to activating transcription factors, most notably those belonging to the ethylene responsive element binding protein family. Interestingly, there were also increased numbers of rosette leaves and plant biomass under nano-ceria exposure, but not under nano-titania. Other transcriptomic responses did not clearly relate to responses observed at the organism level, possibly because of functional and genomic redundancy in Arabidopsis, which may mask expression of morphological changes, despite discernable responses at the transcriptome level. In addition, transcriptomic changes often relate to transgenerational phenotypic development, and hence it may be productive to direct further experimental work to integrate high-throughput genomic results with longer term changes in subsequent generations. Environ Toxicol Chem 2017;36:71-82. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Phenotypic and genomic responses to titanium dioxide and cerium oxide nanoparticles in Arabidopsis germinants.

The effects of exposure to nanoparticles of titanium dioxide (nano-titanium) and cerium oxide (nano-cerium) on gene expression and growth in Arabidopsis thaliana germinants were studied by using microarrays and quantitative real-time polymerase chain reaction (qPCR), and by evaluating germinant phenotypic plasticity. Exposure to 12 d of either nano-titania or nano-ceria altered the regulation of 204 and 142 genes, respectively. Genes induced by the nanoparticles mainly include ontology groups annotated as stimuli responsive, including both abiotic (oxidative stress, salt stress, water transport) and biotic (respiratory burst as a defense against pathogens) stimuli. Further analysis of the differentially expressed genes indicates that both nanoparticles affected a range of metabolic processes (deoxyribonucleic acid [DNA] metabolism, hormone metabolism, tetrapyrrole synthesis, and photosynthesis). Individual exposures to the nanoparticles increased percentages of seeds with emergent radicles, early development of hypocotyls and cotyledons, and those with fully grown leaves. Although there were distinct differences between the nanoparticles in their affect on molecular mechanisms attributable to enhancing germinant growth, both particles altered similar suites of genes related to various pathways and processes related to enhanced growth.

Separate introns gained within short and long soluble peridinin-chlorophyll a-protein genes during radiation of Symbiodinium (Dinophyceae) clade A and B lineages.

Here we document introns in two Symbiodinium clades that were most likely gained following divergence of this genus from other peridinin-containing dinoflagellate lineages. Soluble peridinin-chlorophyll a-proteins (sPCP) occur in short and long forms in different species. Duplication and fusion of short sPCP genes produced long sPCP genes. All short and long sPCP genes characterized to date, including those from free living species and Symbiodinium sp. 203 (clade C/type C2) are intronless. However, we observed that long sPCP genes from two Caribbean Symbiodinium clade B isolates each contained two introns. To test the hypothesis that introns were gained during radiation of clade B, we compared sPCP genomic and cDNA sequences from 13 additional distinct Caribbean and Pacific Symbiodinium clade A, B, and F isolates. Long sPCP genes from all clade B/B1 and B/B19 descendants contain orthologs of both introns. Short sPCP genes from S. pilosum (A/A2) and S. muscatinei (B/B4) plus long sPCP genes from S. microadriaticum (A/A1) and S. kawagutii (F/F1) are intronless. Short sPCP genes of S. microadriaticum have a third unique intron. Symbiodinium clade B long sPCP sequences are useful for assessing divergence among B1 and B19 descendants. Phylogenetic analyses of coding sequences from four dinoflagellate orders indicate that introns were gained independently during radiation of Symbiodinium clades A and B. Long sPCP introns were present in the most recent common ancestor of Symbiodinium clade B core types B1 and B19, which apparently diverged sometime during the Miocene. The clade A short sPCP intron was either gained by S. microadriaticum or possibly by the ancestor of Symbiodinium types A/A1, A3, A4 and A5. The timing of short sPCP intron gain in Symbiodinium clade A is less certain. But, all sPCP introns were gained after fusion of ancestral short sPCP genes, which we confirm as occurring once in dinoflagellate evolution.

Video-rate molecular imaging in vivo with stimulated Raman scattering.

Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and sensitivity compared with magnetic resonance imaging. Stimulated Raman scattering (SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS imaging in living animals and humans has not been feasible because light cannot be collected through thick tissues, and motion-blur arises from slow imaging based on backscattered light. In this work, we enable in vivo SRS imaging by substantially enhancing the collection of the backscattered signal and increasing the imaging speed by three orders of magnitude to video rate. This approach allows label-free in vivo imaging of water, lipid, and protein in skin and mapping of penetration pathways of topically applied drugs in mice and humans.

A composite transcriptional signature differentiates responses towards closely related herbicides in Arabidopsis thaliana and Brassica napus.

In this study, genome-wide expression profiling based on Affymetrix ATH1 arrays was used to identify discriminating responses of Arabidopsis thaliana to five herbicides, which contain active ingredients targeting two different branches of amino acid biosynthesis. One herbicide contained glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), while the other four herbicides contain different acetolactate synthase (ALS) inhibiting compounds. In contrast to the herbicide containing glyphosate, which affected only a few transcripts, many effects of the ALS inhibiting herbicides were revealed based on transcriptional changes related to ribosome biogenesis and translation, secondary metabolism, cell wall modification and growth. The expression pattern of a set of 101 genes provided a specific, composite signature that was distinct from other major stress responses and differentiated among herbicides targeting the same enzyme (ALS) or containing the same chemical class of active ingredient (sulfonylurea). A set of homologous genes could be identified in Brassica napus that exhibited a similar expression pattern and correctly distinguished exposure to the five herbicides. Our results show the ability of a limited number of genes to classify and differentiate responses to closely related herbicides in A. thaliana and B. napus and the transferability of a complex transcriptional signature across species.

Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats.

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.

Evidence for landscape-level, pollen-mediated gene flow from genetically modified creeping bentgrass with CP4 EPSPS as a marker.

Sampling methods and results of a gene flow study are described that will be of interest to plant scientists, evolutionary biologists, ecologists, and stakeholders assessing the environmental safety of transgenic crops. This study documents gene flow on a landscape level from creeping bentgrass (Agrostis stolonifera L.), one of the first wind-pollinated, perennial, and highly outcrossing transgenic crops being developed for commercial use. Most of the gene flow occurred within 2 km in the direction of prevailing winds. The maximal gene flow distances observed were 21 km and 14 km in sentinel and resident plants, respectively, that were located in primarily nonagronomic habitats. The selectable marker used in these studies was the CP4 EPSPS gene derived from Agrobacterium spp. strain CP4 that encodes 5-enol-pyruvylshikimate-3-phosphate synthase and confers resistance to glyphosate herbicide. Evidence for gene flow to 75 of 138 sentinel plants of A. stolonifera and to 29 of 69 resident Agrostis plants was based on seedling progeny survival after spraying with glyphosate in greenhouse assays and positive TraitChek, PCR, and sequencing results. Additional studies are needed to determine whether introgression will occur and whether it will affect the ecological fitness of progeny or the structure of plant communities in which transgenic progeny may become established.

PCP gene family in Symbiodinium from Hippopus hippopus: low levels of concerted evolution, isoform diversity, and spectral tuning of chromophores.

Photosynthetic dinoflagellates have evolved unique water-soluble light harvesting complexes known as peridinin-chlorophyll a-binding proteins (PCPs). Most species of dinoflagellates express either 14 to 17 kDa or 32 to 35 kDa mature PCP apoproteins and do so in stable combinations of isoforms that differ in isoelectric point (pI). The source (posttranslational modification, protein degradation, or genetic) and functional significance of PCP isoform variation have remained unclear. PCPs are encoded by multigene families. However, previous reports conflict over the diversity of PCP genes within gene arrays. We present the first genomic characterization of the PCP gene family from a symbiotic dinoflagellate. Symbiodinium from the Pacific bivalve Hippopus hippopus (203) contains genes for 33 kDa PCP apoproteins that are organized in tandem arrays like those of free-living dinoflagellates Amphidinium carterae, Lingulodinium (Gonyaulax) polyedra, and Heterocapsa pygmaea. The Symbiodinium 203 PCP cassette consists of 1,098-bp coding regions separated by approximately 900-bp spacers. The spacers contain a conserved upstream sequence similar to the promoter in L. polyedra. Surprisingly, sequences of cloned coding regions are not identical, and can differ at up to 2.2% of the nucleotide sites. Sequence variation is found at both silent and nonsilent sites, and analysis of cDNA clones indicate that the variation is present in the mRNA pool. We propose that this variation represents nucleotide diversity among PCP gene copies that are evolving under low-level concerted evolution. Interestingly, the predicted proteins have pIs that are within the range of those published for other species of Symbiodinium. Thus, posttranslational modifications are not necessary to explain the multiple PCP isoforms. We have also identified several polymorphic sites that may influence spectral absorption tuning of chromophores.