Omics reveal distinct mechanisms of toxicity of nanoparticulate and ionic silver in microbes

The mechanisms of toxicity of silver nanoparticles (AgNPs) are not clear and the role of Ag+ released from the nanoparticulated form in the overall toxicity requires further attention, especially in aquatic ecosystems where NPs will most likely end up. The impacts of AgNPs and Ag+ were assessed based on the variations in the overall proteome in i) two aquatic fungal ecotypes of Articulospora tetracladia, one isolated from a non-polluted stream (At72) and the other from a metal-polluted stream (At61), and ii) the bacterial strain Pseudomonas sp. M1 (PsM1) isolated from a metal-polluted stream. Transcriptomic responses of At72 in the same exposure conditions were also assessed to complement knowledge from proteomic responses. At72 was the most sensitive to AgNPs, whereas PsM1 was the most tolerant one. The toxicity of AgNPs was supported by data from NP characterization, which showed increased particle stability and lesser agglomeration in presence of At72 that the other microbes. Omic responses to equitoxic levels of AgNPs and Ag+ suggested different mechanisms of toxicity since distinct profiles of protein and gene expression were unveiled. Gene Ontology (GO) enrichment analysis further unravelled several biological processes and allowed to differentiate the effects of AgNPs from those of Ag+. Overall, omic approaches revealed different adaptive responses to Ag+ or AgNPs in the metabolic, energetic and stress pathways. Furthermore, the negligible amount of Ag+ released from AgNPs suggested that toxicity of AgNPs was mainly associated with the particle for

Proteomic responses to nanoparticulate and ionic silver in freshwater microbes with different background

ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by the Portuguese Foundation for Science and Technology (FCT) I.P. through the strategic funding UID/BIA/04050/2014 (POCI-01-0145-FEDER-007569) and PTDC/AAC-AMB/121650/201

Proteomics and antioxidant enzymes reveal different mechanisms of toxicity induced by ionic and nanoparticulate silver in bacteria

The increased use of silver nanoparticles (AgNPs) raises concerns about their impacts on aquatic ecosystems. The impacts of Ag+ and AgNPs were assessed on proteomic and antioxidant enzymatic responses of Pseudomonas sp. M1. The effects of Ag+ on bacterial growth were stronger than those of AgNPs (EC20 = 107.1 μg L−1 for Ag+; EC20 = 307.2 μg L−1 for AgNPs), indicating the lower toxicity of the latter. At EC20, the activities of antioxidant enzymes increased more under exposure to Ag+ than to AgNPs, particularly for superoxide dismutase and glutathione peroxidase (stimulation of 667% and 433%, respectively). A total of 166 proteins were identified by SWATH-MS; among these, only 59 had their content significantly altered by one or both forms of silver. Exposure to AgNPs resulted in an increase of about 54% of these proteins, whereas 54% decreased under exposure to Ag+. Gene Ontology enrichment analysis revealed that protein folding and transmembrane transport were the most relevant processes affected by Ag+ exposure, whereas AgNPs mostly affected translation. Also, results suggest that each form of silver induced different adaptive responses. Furthermore, the low levels of Ag+ released from AgNPs (<0.1%) support a minor role of dissolved silver in AgNP toxicity to Pseudomonas sp. M1ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by the Portuguese Foundation for Science and Technology I.P. (FCT) through the strategic funding UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), PTDC/AAC-AMB/121650/2010, POCI-01-0145-FEDER-007440 (ref. UID/NEU/04539/2013), the National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER402-022125 (ref. ROTEIRO/0028/2013), and PTDC/BIA-BMA/ 30922/201

Intraspecific traits change biodiversity effects on ecosystem functioning under metal stress

The online version of this article (doi:10.1007/s00442-011-1930-3) contains supplementary material, which is available to authorized users.Studies investigating the impacts of biodiversity loss on ecosystem processes have often reached different conclusions, probably because insufficient attention has been paid to some aspects including (1) which biodiversity measure (e.g., species number, species identity or trait) better explains ecosystem functioning, (2) the mechanisms underpinning biodiversity effects, and (3) how can environmental context modulates biodiversity effects. Here, we investigated how species number (one to three species) and traits of aquatic fungal decomposers (by replacement of a functional type from an unpolluted site by another from a metal-polluted site) affect fungal production (biomass acumulation) and plant litter decomposition in the presence and absence of metal stress. To examine the putative mechanisms that explain biodiversity effects, we determined the contribution of each fungal species to the total biomass produced in multicultures by real-time PCR. In the absence of metal, positive diversity effects were observed for fungal production and leaf decomposition as a result of species complementarity. Metal stress decreased diversity effects on leaf decomposition in assemblages containing the functional type from the unpolluted site, probably due to competitive interactions between fungi. However, dominance effect maintained positive diversity effects under metal stress in assemblages containing the functional type from the metal-polluted site. These findings emphasize the importance of intraspecific diversity in modulating diversity effects under metal stress, providing evidence that trait-based diversity measures should be incorporated when examining biodiversity effects.The Portuguese Foundation for Science and Technology supported I. Fernandes (SFRH/BD/42215/2007

Effects of riparian plant diversity loss on aquatic microbial decomposers become more pronounced at longer times

We examined the potential long-term impacts of riparian plant diversity loss on diversity and activity of aquatic microbial decomposers. Microbial assemblages were obtained in a mixed-forest stream by immersion of mesh bags contain-ing three leaf species (alder, oak and eucalyptus), commonly found in riparian corridors of Iberian streams. Simulation of species loss was done in microcosms by including a set of all leaf species, retrieved from the stream, and non-colonized leaves of three, two or one leaf species. Leaves were renewed every month throughout six months, and microbial inoculum was ensured by a set of colonized leaves from the previous month. Microbial diversity, leaf mass loss and fungal biomass were assessed at the second and sixth months after plant species loss. Molecular diversity of fungi and bacteria, as the total number of operational taxonomic units per leaf diversity treatment, decreased with leaf diversity loss. Fungal biomass tended to decrease linearly with leaf species loss on oak and eucalyptus, suggesting more pronounced effects of leaf diver-sity on lower quality leaves. Decomposition of alder and eucalyptus leaves was affected by leaf species identity, mainly after longer times following diversity loss. Leaf decomposi-tion of alder decreased when mixed with eucalyptus, while decomposition of eucalyptus decreased in mixtures with oak. Results suggest that the effects of leaf diversity on microbial decomposers depended on leaf species number and also on which species were lost from the system, especially after longer times. This may have implications for the management of riparian forests to maintain stream ecosystem functioning.FEDER-POFC-COMPETE and the Portuguese Foundation for Science and Technology supported this study (PEst-C/ BIA/UI4050/2011, PTDC/AAC-AMB/113746/2009 and PTDC/AAC-AMB/117068/2010), S. Duarte (SFRH/BPD/47574/2008) and I. Fernandes (SFRH/BD/42215/2007)

The role of the freshwater shrimp atyaephyra desmarestii in leaf litter breakdown in streams

This version does not correspond to the published one. To access the final version go to: http://www.springerlink.com/content/a31518u452m03286/In aquatic ecosystems, microorganisms and invertebrates provide critical links between plant detritus and higher trophic levels. Atyaephyra desmarestii is an omnivorous decapod that inhabits freshwaters and exhibits high tolerance to temperature oscillations and high ability to colonize new habitats. Although A. desmarestii is able to ingest a variety of foods, few studies have been conducted to elucidate the role of this freshwater shrimp on detritus breakdown in streams. In this study, A. desmarestii was allowed to feed on conditioned or unconditioned alder and eucalyptus leaves in microcosms with or without access to its fecal pellets. At the end of the experiment, total body length of the animals was measured, and the remaining leaves and fecal pellets were used for dry mass quantification and assessment of bacterial and fungal diversity by denaturing gradient gel electrophoresis (DGGE). Cluster analyses of DGGE fingerprints indicated that the major differences in microbial communities on leaves were between leaf types, while on fecal pellets were between conditioned and unconditioned leaves. However, the consumption rate by the shrimp did not differ between leaf types, and was significantly higher on leaves conditioned by microorganisms and in treatments without access to feces. In treatments without access to feces, the production of feces and fine particulate organic matter was also significantly higher for conditioned leaves. Overall, our results support the feeding plasticity of A. desmarestii and its potential role in plant litter breakdown in streams. This might have implications for maintaining stream ecosystem functioning, particularly if more vulnerable shredders decline.The Portuguese Foundation for the Science and Technology supported S. Duarte (SFRH/BPD/47574/2008

Effects of cadmium and phenanthrene mixtures on aquatic fungi and microbially mediated leaf litter decomposition

This version does not correspond to the published one. To access the final version go to: http://www.springerlink.com/content/t8t302617003m078/Urbanization and industrial activities have contributed to widespread contamination by metals and polycyclic aromatic hydrocarbons, but the combined effects of these toxics on aquatic biota and processes are poorly understood. We examined the effects of cadmium (Cd) and phenanthrene on the activity and diversity of fungi associated with decomposing leaf litter in streams. Leaves of Alnus glutinosa were immersed for 10 days in an unpolluted low-order stream in northwest Portugal to allow microbial colonization. Leaves were then exposed in microcosms for 14 days to Cd (0.06–4.5 mg L−1) and phenanthrene (0.2 mg L−1) either alone or in mixture. A total of 19 aquatic hyphomycete species were found sporulating on leaves during the whole study. The dominant species was Articulospora tetracladia, followed by Alatospora pulchella, Clavatospora longibrachiata, and Tetrachaetum elegans. Exposure to Cd and phenanthrene decreased the contribution of A. tetracladia to the total conidial production, whereas it increased that of A. pulchella. Fungal diversity, assessed as denaturing gradient gel electrophoresis fingerprinting or conidial morphology, was decreased by the exposure to Cd and/or phenanthrene. Moreover, increased Cd concentrations decreased leaf decomposition and fungal reproduction but did not inhibit fungal biomass production. Exposure to phenanthrene potentiated the negative effects of Cd on fungal diversity and activity, suggesting that the co-occurrence of these stressors may pose additional risk to aquatic biodiversity and stream ecosystem functioning.The Portuguese Foundation for the Science and Technology supported this work (POCI/MAR/56964/2004) and S. Duarte (SFRH/BPD/47574/2008

Intraspecific Variation of the Aquatic Fungus Articulospora tetracladia: An Ubiquitous Perspective

The worldwide-distributed aquatic fungus Articulospora tetracladia Ingold is a dominant sporulating species in streams of the Northwest Iberian Peninsula. To elucidate the genetic diversity of A. tetracladia, we analyzed isolates collected from various types of plant litter or foam in streams from North and Central Portugal and North Spain, between 2000 and 2010. Genetic diversity of these fungal populations was assessed by denaturing gradient gel electrophoresis (DGGE) fingerprints and by using ITS1-5.8S-ITS2 barcodes. Moreover, ITS1-5.8S-ITS2 barcodes of A. tetracladia reported in other parts of the world (Central Europe, United Kingdom, Canada, Japan and Malaysia) were retrieved from the National Center for Biotechnology (NCBI) and the National Institute of Technology and Evaluation Biological Resource Center (NBRC) to probe into genetic diversity of A. tetracladia. PCR-DGGE of ITS2 region of 50 Iberian fungal isolates distinguished eight operational taxonomic units (OTUs), which were similar to those obtained from neighboring trees based on ITS2 gene sequences. On the other hand, ITS1-5.8S-ITS2 barcodes of 68 fungal isolates yielded nine OTUs, but five fungal isolates were not assigned to any of these OTUs. Molecular diversity was highest for OTU-8, which included only European isolates. Two haplotypes were observed within OTU-8 and OTU-9, while only one haplotype was found within each of the remaining OTUs. Malaysia did not share haplotypes with other countries. Overall results indicate that, apart from the Malaysian genotypes, A. tetracladia genotypes were geographically widespread irrespective of sampling time, sites or substrates. Furthermore, PCR-DGGE appeared to be a rapid tool for assessing intraspecific diversity of aquatic hyphomycetes

Taxa-area relationship of aquatic fungi on deciduous leaves

One of the fundamental patterns in macroecology is the increase in the number of observed taxa with size of sampled area. For microbes, the shape of this relationship remains less clear. The current study assessed the diversity of aquatic fungi, by the traditional approach based on conidial morphology (captures reproducing aquatic hyphomycetes) and next generation sequencing (NGS; captures other fungi as well), on graded sizes of alder leaves (0.6 to 13.6 cm2). Leaves were submerged in two streams in geographically distant locations: the Oliveira Stream in Portugal and the Boss Brook in Canada. Decay rates of alder leaves and fungal sporulation rates did not differ between streams. Fungal biomass was higher in Boss Brook than in Oliveira Stream, and in both streams almost 100% of the reads belonged to active fungal taxa. In general, larger leaf areas tended to harbour more fungi, but these findings were not consistent between techniques. Morphospecies-based diversity increased with leaf area in Boss Brook, but not in Oliveira Stream; metabarcoding data showed an opposite trend. The higher resolution of metabarcoding resulted in steeper taxa-accumulation curves than morphospecies-based assessments (fungal conidia morphology). Fungal communities assessed by metabarcoding were spatially structured by leaf area in both streams. Metabarcoding promises greater resolution to assess biodiversity patterns in aquatic fungi and may be more accurate for assessing taxa-area relationships and local to global diversity ratios.This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), funded by national funds through the Portuguese Foundation for Science and Technology (FCT) I.P. (http://www.fct.pt/) and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI) and by the project PTDC/AAC-AMB/117068/2010, funded by national funds through FCT I.P. and the European Regional Development Funds through the Operational Competitiveness Program (FEDER-COMPETE). Support from FCT to SD (SFRH/BPD/47574/2008 and SFRH/BPD/109842/2015) and from NSERC Discovery grant program (http://www.nserc-crsng.gc.ca/index_eng.asp) to FB is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio