Interactions shape aquatic microbiome responses to Cu and Au nanoparticle treatments in wetland manipulation experiments.

In natural systems, organisms are embedded in complex networks where their physiology and community composition is shaped by both biotic and abiotic factors. Therefore, to assess the ecosystem-level effects of contaminants, we must pair complex, multi-trophic field studies with more targeted hypothesis-driven approaches to explore specific actors and mechanisms. Here, we examine aquatic microbiome responses to long-term additions of commercially-available metallic nanoparticles [copper-based (CuNPs) or gold (AuNPs)] and/or nutrients in complex, wetland mesocosms over 9 months, allowing for a full growth cycle of the aquatic plants. We found that both CuNPs and AuNPs (but not nutrient) treatments showed shifts in microbial communities and populations largely at the end of the experiment, as the aquatic plant community senesced. we examine aquatic microbiomes under chronic dosing of NPs and nutrients Simplified microbe-only or microbe + plant incubations revealed that direct effects of AuNPs on aquatic microbiomes can be buffered by plants (regardless of seasonal As mesocosms were dosed weekly, the absence of water column accumulation indicates the partitioning of both metals into other environmental compartments, mainly the floc and aquatic plants photosynthetically-derived organic matter. Overall, this study identifies the potential for NP environmental impacts to be either suppressed by or propagated across trophic levels via the presence of primary producers, highlighting the importance of organismal interactions in mediating emerging contaminants' ecosystem-wide impacts.

Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

Seasonal Differences and Grazing Pressure Alter the Fate of Gold Nanoparticles in a Microcosm Experiment.

Gold nanoparticles (AuNPs) are used as models to track and predict NP fates and effects in ecosystems. Previous work found that aquatic macrophytes and their associated biofilm primarily drove the fate of AuNPs within aquatic ecosystems and that seasonality was an important abiotic factor in the fate of AuNPs. Therefore, the present work aims to study if grazers, by feeding on these interfaces, modify the AuNP fate and if this is altered by seasonal fluctuations. Microcosms were dosed with 44.8 µg/L of AuNP weekly for 4 weeks and maintained in environmental chambers simulating Spring and Fall light and temperature conditions. We discovered that seasonal changes and the presence of grazers significantly altered the fate of Au. Higher temperatures in the warmer season increased dissolved organic carbon (DOC) content in the water column, leading to stabilization of Au in the water column. Additionally, snail grazing on biofilm growing on the Egeria densa surface led to a transfer of Au from macrophytes to the organic matter above the sediments. These results demonstrate that climate and grazers significantly impacted the fate of Au from AuNPs, highlighting the role that grazers might have in a large and biologically more complex ecosystem.

Xenobiotic metabolism and transport in Caenorhabditis elegans.

Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this species has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolation to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and the role of the worm microbiome in xenobiotic transformation were described. Then a review of what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was performed. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.

Long-Term Effects of Copper Nanopesticides on Soil and Sediment Community Diversity in Two Outdoor Mesocosm Experiments.

The use of novel pesticides containing nanomaterials (nanopesticides) is growing and is considered a promising approach to reduce the impacts of agriculture on the environment and human health. However, the environmental effects of these novel agrochemicals are not fully characterized, and more research is needed to determine the benefits and risks they confer. Here, we assessed the impacts of repeated exposures to a Cu(OH)2 nanopesticide on the soil and sediment biodiversity of target (terrestrial) and nontarget (wetland) ecosystems by performing long-term outdoor mesocosm experiments. As pesticides are often used concomitantly with other agrochemicals, we also tested for interactive effects between nanopesticide exposure and fertilization treatments in both ecosystems. We used high-throughput sequencing on three marker genes to characterize effects on bacterial, fungal, and total eukaryotic community structure and diversity. Interestingly, we found limited effects of nanopesticide exposure on the terrestrial soil communities. Conversely, we found significant shifts in the sediment communities of the wetland mesocosms, especially for eukaryotes (protists, fungi, and algae). In the absence of fertilization, fungal and total eukaryotic community compositions exposed to nanopesticides for long periods of time were distinct from unexposed communities. We identified 60 taxa that were significantly affected by nanopesticide exposure, most of which were microeukaryotes affiliated to cercozoans, Gastrotricha, or unicellular algal taxa. Our study suggests that this nanopesticide has limited effects on the soil biodiversity of a target terrestrial agroecosystem, while nontarget aquatic communities are more sensitive, particularly among protists which are not targeted by this bactericide/fungicide.

Copper and Gold Nanoparticles Increase Nutrient Excretion Rates of Primary Consumers.

Freshwater ecosystems are exposed to engineered nanoparticles through municipal and industrial wastewater-effluent discharges and agricultural nonpoint source runoff. Because previous work has shown that engineered nanoparticles from these sources can accumulate in freshwater algal assemblages, we hypothesized that nanoparticles may affect the biology of primary consumers by altering the processing of two critical nutrients associated with growth and survivorship, nitrogen and phosphorus. We tested this hypothesis by measuring the excretion rates of nitrogen and phosphorus of Physella acuta, a ubiquitous pulmonate snail that grazes heavily on periphyton, exposed to either copper or gold engineered nanoparticles for 6 months in an outdoor wetland mesocosm experiment. Chronic nanoparticle exposure doubled nutrient excretion when compared to the control. Gold nanoparticles increased nitrogen and phosphorus excretion rates more than copper nanoparticles, but overall, both nanoparticles led to higher consumer excretion, despite contrasting particle stability and physiochemical properties. Snails in mesocosms enriched with nitrogen and phosphorus had overall higher excretion rates than ones in ambient (no nutrients added) mesocosms. Stimulation patterns were different between nitrogen and phosphorus excretion, which could have implications for the resulting nutrient ratio in the water column. These results suggest that low concentrations of engineered nanoparticles could alter the metabolism of consumers and increase consumer-mediated nutrient recycling rates, potentially intensifying eutrophication in aquatic systems, for example, the increased persistence of algal blooms as observed in our mesocosm experiment.

Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment.

Despite the rapid rise in diversity and quantities of engineered nanomaterials produced, the impacts of these emerging contaminants on the structure and function of ecosystems have received little attention from ecologists. Moreover, little is known about how manufactured nanomaterials may interact with nutrient pollution in altering ecosystem productivity, despite the recognition that eutrophication is the primary water quality issue in freshwater ecosystems worldwide. In this study, we asked two main questions: (1) To what extent do manufactured nanoparticles affect the biomass and productivity of primary producers in wetland ecosystems? (2) How are these impacts mediated by nutrient pollution? To address these questions, we examined the impacts of a citrate-coated gold nanoparticle (AuNPs) and of a commercial pesticide containing Cu(OH)2 nanoparticles (CuNPs) on aquatic primary producers under both ambient and enriched nutrient conditions. Wetland mesocosms were exposed repeatedly with low concentrations of nanoparticles and nutrients over the course of a 9-month experiment in an effort to replicate realistic field exposure scenarios. In the absence of nutrient enrichment, there were no persistent effects of AuNPs or CuNPs on primary producers or ecosystem productivity. However, when combined with nutrient enrichment, both NPs intensified eutrophication. When either of these NPs were added in combination with nutrients, algal blooms persisted for >50 d longer than in the nutrient-only treatment. In the AuNP treatment, this shift from clear waters to turbid waters led to large declines in both macrophyte growth and rates of ecosystem gross primary productivity (average reduction of 52% ± 6% and 92% ± 5%, respectively) during the summer. Our results suggest that nutrient status greatly influences the ecosystem-scale impact of two emerging contaminants and that synthetic chemicals may be playing an under-appreciated role in the global trends of increasing eutrophication. We provide evidence here that chronic exposure to Au and Cu(OH)2 nanoparticles at low concentrations can intensify eutrophication of wetlands and promote the occurrence of algal blooms.

Comprehensive characterization of mitochondrial bioenergetics at different larval stages reveals novel insights about the developmental metabolism of Caenorhabditis elegans.

Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. Caenorhabditis elegans is widely used to study developmental biology, mitochondrial disease, and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species, and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic, short-term responses including arrested development and death, and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans. We optimized and describe conditions for analysis of basal OCR, basal mitochondrial OCR, ATP-linked OCR, spare and maximal respiratory capacity, proton leak, and non-mitochondrial OCR. A key consideration is normalization, and we present and discuss results as normalized per individual worm, protein content, worm volume, mitochondrial DNA (mtDNA) count, nuclear DNA (ncDNA) count, and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked, and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans. Broadly, when normalized to worm number, our results agree with previous reports in showing dramatic increases in OCR throughout development. However, when normalized to total protein, worm volume, or ncDNA or mtDNA count, after a significant 2-3-fold increase from L1 to L2 stages, we found small or no changes in most OCR parameters from the L2 to the L4 stage, other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall, our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.

Rotenone Modulates Caenorhabditis elegans Immunometabolism and Pathogen Susceptibility.

Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Exposures to these mitotoxicants may pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. In this study, we investigated whether the Complex I inhibitor rotenone could alter C. elegans immunometabolism and disease susceptibility. C. elegans embryos were exposed to rotenone (0.5 µM) or DMSO (0.125%) until they reached the L4 larval stage. Inhibition of mitochondrial respiration by rotenone and disruption of mitochondrial metabolism were evidenced by rotenone-induced detrimental effects on mitochondrial efficiency and nematode growth and development. Next, through transcriptomic analysis, we investigated if this specific but mild mitochondrial stress that we detected would lead to the modulation of immunometabolic pathways. We found 179 differentially expressed genes (DEG), which were mostly involved in detoxification, energy metabolism, and pathogen defense. Interestingly, among the down-regulated DEG, most of the known genes were involved in immune defense, and most of these were identified as commonly upregulated during P. aeruginosa infection. Furthermore, rotenone increased susceptibility to the pathogen Pseudomonas aeruginosa (PA14). However, it increased resistance to Salmonella enterica (SL1344). To shed light on potential mechanisms related to these divergent effects on pathogen resistance, we assessed the activation of the mitochondrial unfolded protein response (UPRmt), a well-known immunometabolic pathway in C. elegans which links mitochondria and immunity and provides resistance to pathogen infection. The UPRmt pathway was activated in rotenone-treated nematodes further exposed for 24 h to the pathogenic bacteria P. aeruginosa and S. enterica or the common bacterial food source Escherichia coli (OP50). However, P. aeruginosa alone suppressed UPRmt activation and rotenone treatment rescued its activation only to the level of DMSO-exposed nematodes fed with E. coli. Module-weighted annotation bioinformatics analysis was also consistent with UPRmt activation in rotenone-exposed nematodes consistent with the UPR being involved in the increased resistance to S. enterica. Together, our results demonstrate that the mitotoxicant rotenone can disrupt C. elegans immunometabolism in ways likely protective against some pathogen species but sensitizing against others.

Plant and Microbial Responses to Repeated Cu(OH)2 Nanopesticide Exposures Under Different Fertilization Levels in an Agro-Ecosystem.

The environmental fate and potential impacts of nanopesticides on agroecosystems under realistic agricultural conditions are poorly understood. As a result, the benefits and risks of these novel formulations compared to the conventional products are currently unclear. Here, we examined the effects of repeated realistic exposures of the Cu(OH)2 nanopesticide, Kocide 3000, on simulated agricultural pastureland in an outdoor mesocosm experiment over 1 year. The Kocide applications were performed alongside three different mineral fertilization levels (Ambient, Low, and High) to assess the environmental impacts of this nanopesticide under low-input or conventional farming scenarios. The effects of Kocide over time were monitored on forage biomass, plant mineral nutrient content, plant-associated non-target microorganisms (i.e., N-fixing bacteria or mycorrhizal fungi) and six soil microbial enzyme activities. We observed that three sequential Kocide applications had no negative effects on forage biomass, root mycorrhizal colonization or soil nitrogen fixation rates. In the Low and High fertilization treatments, we observed a significant increase in aboveground plant biomass after the second Kocide exposure (+14% and +27%, respectively). Soil microbial enzyme activities were significantly reduced in the short-term after the first exposure (day 15) in the Ambient (-28% to -82%) and Low fertilization (-25% to -47%) but not in the High fertilization treatment. However, 2 months later, enzyme activities were similar across treatments and were either unresponsive or responded positively to subsequent Kocide additions. There appeared to be some long-term effects of Kocide exposure, as 6 months after the last Kocide exposure (day 365), both beta-glucosidase (-57% in Ambient and -40% in High fertilization) and phosphatase activities (-47% in Ambient fertilization) were significantly reduced in the mesocosms exposed to the nanopesticide. These results suggest that when used in conventional farming with high fertilization rates, Kocide applications did not lead to marked adverse effects on forage biomass production and key plant-microorganism interactions over a growing season. However, in the context of low-input organic farming for which this nanopesticide is approved, Kocide applications may have some unintended detrimental effects on microbially mediated soil processes involved in carbon and phosphorus cycling.