Does functionalised nanoplastics modulate the cellular and physiological responses of aquatic fungi to metals?

Co-contamination of freshwaters by nanoplastics (NPs; ≤ 1 µm) and metals is an emerging concern. Aquatic hyphomycetes play a crucial role as primary decomposers in these ecosystems. However, concurrent impacts of NPs and metals on the cellular and physiological activities of these fungi remain poorly understood. Here, the effects of environmentally realistic concentrations of two types of polystyrene (PS) NPs (bare and -COOH; up to 25 µg L-1) and copper (Cu; up to 50 µg L-1) individually and all possible combinations (NPs types and Cu) on Articulospora tetracladia, a prevalent aquatic hyphomycete, were investigated. Endpoints measured were intracellular reactive oxygen species accumulation, plasma membrane disruption and fungal growth. The results suggest that functionalised (-COOH) NPs enhance Cu adsorption, as revealed by spectroscopic analyses. Notably, NPs, Cu and their co-exposure to A. tetracladia can lead to ROS accumulation and plasma membrane disruption. In most cases, exposure to treatments containing -COOH NPs with Cu showed greater cellular response and suppressed fungal growth. By contrast, exposure to Cu individually showed stimulatory effects on fungal growth. Overall, this study provides novel insight that functionalisation of NPs facilitates metal adsorption, thus modulating the impacts of metals on aquatic fungi.

Advances in the design of amino acid and peptide synthesized gold nanoparticles for their applications.

The field of therapeutics and diagnostics is advanced by nanotechnology-based approaches including the spatial-temporal release of drugs, targeted delivery, enhanced accumulation of drugs, immunomodulation, antimicrobial action, and high-resolution bioimaging, sensors and detection. Various compositions of nanoparticles (NPs) have been developed for biomedical applications; however, gold NPs (Au NPs) have attracted tremendous attention due to their biocompatibility, easy surface functionalization and quantification. Amino acids and peptides have natural biological activities as such, their activities enhance several folds in combination with NPs. Although peptides are extensively used to produce various functionalities of Au NPs, amino acids have also gained similar interests in producing amino acid-capped Au NPs due to the availability of amine, carboxyl and thiol functional groups. Henceforth, a comprehensive review is needed to timely bridge the synthesis and the applications of amino acid and peptide-capped Au NPs. This review aims to describe the synthesis mechanism of Au NPs using amino acids and peptides along with their applications in antimicrobial, bio/chemo-sensors, bioimaging, cancer therapy, catalysis, and skin regeneration. Moreover, the mechanisms of various activities of amino acid and peptide capped-Au NPs are presented. We believe this review will motivate researchers to better understand the interactions and long-term activities of amino acid and peptide-capped Au NPs for their success in various applications.

Acidic sophorolipid and antimicrobial peptide based formulation as antimicrobial and antibiofilm agents.

Antimicrobial peptides (AMPs) are considered promising candidates to treat various infections in soft tissues and skin. However, no effective treatment based on AMPs has been reached to clinics due to their instability in serum and wounds. Biosurfactants such as acidic sophorolipids (ASLs) of very high concentrations (equal or above 5 mg/mL) have been demonstrated to be antimicrobial agents, however these concentrations might induce cytotoxic effects to human cells. Here, we have demonstrated the synergistic antimicrobial effect of ASL nanoparticles (NPs) and LL37 peptides (below their minimum inhibitory concentrations; MICs) to eradicate Gram-positive and Gram-negative bacteria in human serum (HS) and in the presence of trypsin. The formulations containing ASL NPs (500 µg/mL) and LL37 peptides (15-25 µg/mL) effectively kill wide strains of bacteria in 5 % HS and the presence of trypsin. Moreover, the combination of ASL NPs (500 µg/mL) and LL37 peptides (15 µg/mL) prevents the formation of S. aureus biofilm and eradicates the one-day old biofilm. Importantly, the combination of ASL NPs and LL37 peptides severely damages the cell membrane of Escherichia coli (E. coli) as shown by atomic force microscopy (AFM). The combination of ASL NPs and LL37 peptides rapidly damages the outer (OM) and inner membrane (IM) of E. coli, while ASL NPs (1000 µg/mL) alone slowly compromise the integrity of the bacterial membrane. Importantly, the combination of ASL NPs and LL37 peptides is biocompatible to human keratinocyte cells (HaCaTs) and human umbilical vein endothelial cells (HUVECs), and induces the expression of anti-inflammatory cytokine in macrophages. Overall, ASL NPs in combination with LL37 peptides might be developed as an effective topical formulation to prevent bacterial infections in the skin.

Impacts of low concentrations of nanoplastics on leaf litter decomposition and food quality for detritivores in streams.

In forested streams, leaf litter decomposition is a vital ecosystem process, governed primarily by aquatic hyphomycetes. These fungi are crucial mediators of nutrients and energy to invertebrates and higher trophic levels. Very little information is available on the impact of low concentrations of different sizes of nanoplastic particles (NPPs) on leaf litter decomposition and aquatic hyphomycetes communities. Besides, NPPs impact on leaf litter nutritional quality and invertebrate feeding behaviour is unknown. We conducted a microcosm assay with varying concentrations (0-25 µg L-1) of small (100 nm; SNPPs) and large (1000 nm; LNPPs) plastic particles to assess their impact on leaf litter decomposition, sporulation rates and community structure of aquatic hyphomycetes. Furthermore, leaf litter was retrieved and fed to invertebrates to assess feeding rates. Our results indicated that leaf litter decomposition, fungal sporulation and abundance were significantly affected by NPPs concentrations and sizes. By contrast, leaf litter nutritional quality was impacted only by sizes. The NPPs, particularly SNPPs, augmented leaf litter polyunsaturated fatty acids (18-31%), consequently improving food quality; however, invertebrates' feeding rates were not impacted. Overall, our study provides novel insights on the risks posed by NPPs with pronounced impact at the basal trophic level.

Plastisphere in freshwaters: An emerging concern.

Plastisphere, an ecosystem of microbes thriving on floating plastic debris, has been extensively studied in marine waters since 2013. Currently, very little is known about the freshwater plastisphere. This review seeks to provide a broad insight into the freshwater science of plastisphere in the light of marine plastisphere, including research gaps, suggestions, and rising concerns, which would be of interest to the public, policymakers, and stakeholders. Given that freshwaters are endangered ecosystems, it is imperative to understand the role and impact of plastisphere on freshwaters. Plastic debris, especially microplastics (size <5 mm) in freshwater ecosystems, provide a stable, persistent, and buoyant substrate for microbes. Although current evidence suggests that freshwater environmental conditions and microplastics' physical and chemical properties significantly influence microbial colonisation, its role and integration in the aquatic ecosystems are unknown. Considering that the plastisphere biodiversity is unique, we seek to establish why and how many species co-exist in the plastisphere. Evaluating such fundamental questions should advance our basic understanding of the resilience of plastisphere to the changing environment. Plastisphere microbes, including the pathogenic bacteria, were found in both systems demonstrating their ability to survive on the plastic fragments from one ecosystem to another. A significant concern regarding plastisphere is the potential freshwater dispersal of anthropogenic pollutants and invasive or pathogenic species. Notably, microplastics aggregates may serve as a food source for grazers, which opens the question of the extent to which it can impact freshwater food webs. To gain a thorough understanding of the interplay between microplastics and the biogeochemical cycle, further insight into plastisphere microbes' functional role is needed. This would shed light on the unconsidered freshwater elemental cycling pathways. Given the complexity and universal nature of the plastisphere, strong interdisciplinary global research initiatives or networks are required to address the emerging concerns of plastisphere in freshwaters.

Metabolomic, functional, and ecologic responses of the common freshwater fungus Neonectria lugdunensis to mine drainage stress.

Metal contamination of watersheds is a global problem. Here, we conducted litter decomposition studies with Neonectria lugdunensis, a cosmopolitan aquatic fungus. Fungal isolates from four reference (non-impacted) and six metal-contaminated streams (due to mine drainage) were exposed to mine drainage and reference stream waters in Central Portugal. Impact of mine drainage waters on N. lugdunensis hyphae was investigated by performing metabolomic profiling of 200 lipids and 25 amino acids (AA) with ultra-high performance liquid chromatography-mass spectrometry. In parallel, functional response of N. lugdunensis isolates was assessed through expression profiles of a functional gene, cellobiohydrolase I (CbhI). Ecological performance via leaf mass loss was also determined. Exposure to mine drainage waters altered the concentration of numerous AA and lipids. Most strikingly, a gradual increase in the concentration of the triacylglycerols (TAG) with shorter acyl chains and lesser unsaturation was observed after the exposure to mine drainage waters. In addition, the changes in the concentration of numerous TAG, lysophosphatidylcholines, and AA were more significant in the isolates from the metal-contaminated streams after exposure to mine drainage water. CbhI gene of the isolates from reference streams was down-regulated by metal stress, while those from metal-contaminated streams remained unaffected. Finally, leaf mass loss was influenced by both exposure to mine drainage waters and the origin of isolates. Overall, our study demonstrates unique functional signatures displayed by fungi under metal stress and the relevant role that fungal AA and lipids play to cope with metal toxicity.

Inter- and intraspecific functional variability of aquatic fungal decomposers and freshwater ecosystem processes.

Although considerable intraspecific trait variation is common, research dedicated to ecosystem functioning has focused mainly on species diversity. Organic matter breakdown, a key ecosystem-level process in woodland streams is mainly driven by aquatic hyphomycetes. These aquatic fungal decomposers constitute a critical link between plant litter and invertebrate detritivores in detritus-based food webs in streams. In this study, we evaluated the functional variability across a set of ten isolates each belonging to five widespread aquatic hyphomycete species, namely Articulospora tetracladia, Anguillospora crassa, Lemonniera terrestris, Neonectria lugdunensis and Tetracladium marchalianum. All the isolates originated from undisturbed streams. We estimated inter- and intraspecific variability on growth rates, litter decomposition and sporulation rates of the isolates. In addition, we also assessed the invertebrate consumption rates on leaves colonized by different isolates. Significant differences were observed within the fungal species in growth rates (A. crassa, L. terrestris, N. lugdunensis and T. marchalianum), leaf litter decomposition (A. tetracladia, L. terrestris and N. lugdunensis) and sporulation rates (A. crassa, A. tetracladia, L. terrestris and N. lugdunensis). The relative consumption rates of the shredder Schizopelex festiva significantly differed when fed with leaves colonized by isolates of L. terrestris and N. lugdunensis, however differences were not seen when fed with leaves conditioned by different species. Overall, results indicate substantial intraspecific functional variability among the isolates of aquatic hyphomycetes. Besides, our study also provides a novel insight that intraspecific functional variability is a natural phenomenon exhibited by isolates not only from polluted but also from undisturbed streams. Here the isolates demonstrated marked inter- and intraspecific functional variability, calling for a greater understanding of the functional role of aquatic hyphomycetes and its ability to influence higher trophic levels.

Short-term exposure to low concentrations of copper oxide nanoparticles can negatively impact the ecological performance of a cosmopolitan freshwater fungus.

In the past few decades, the use of nano-based products has been expanding, subsequently increasing the risk of aquatic contamination by nanoparticles (NPs). Consequently, it is important to assess the potential risks of environmentally realistic concentrations of NPs. Leaf litter decomposition, a fundamental ecological process in headwater streams, is governed mainly by fungi. In this study, copper oxide nanoparticles (CuO NPs) were synthesised, and the influence of short term exposure to low concentrations of copper oxide nanoparticles on leaf litter decomposition and fungal reproduction (sporulation) was assessed. A cosmopolitan aquatic fungal decomposer species Articulospora tetracladia was used to assess litter decomposition upon exposure to CuO NPs (0.00, 0.04, 0.08 and 0.12 mg L-1) for a short time period (1, 2, 3 and 4 days). Concentrations and exposure time of NPs affected both leaf litter decomposition and sporulation. A significant difference in leaf litter decomposition and fungal sporulation was observed between the control (0.0 mg L-1) and all concentrations tested but not between 0.04, 0.08 and 0.12 mg L-1 of NPs. All the exposure time points (1, 2, 3 and 4 days) had a significant impact on sporulation; however a significant difference in leaf litter mass loss was seen between all time points except for between 2 and 3 days. Overall, our study provides a novel insight into the potential risk of short-term exposure to environmentally realistic concentrations of CuO NPs on the ecological performance of a cosmopolitan freshwater fungus.

Can low concentrations of metal oxide and Ag loaded metal oxide nanoparticles pose a risk to stream plant litter microbial decomposers?

Nanoparticles (NPs) continue to be extensively produced by many industries, which ultimately leads to its release into the aquatic environment. It is crucial to estimate the impact of low concentrations of NPs which are environmentally relevant. Litter decomposition is a key ecological process in forested streams; microbes like fungi and bacteria are recognised to play a vital role in litter decomposition. In this study, zinc oxide (ZnO), titanium dioxide (TiO2), silver loaded ZnO (Ag/ZnO) and silver loaded TiO2 (Ag/TiO2) NPs were synthesized, and impacts of low concentrations (0, 2.5, 25, 50 µM) on leaf litter decomposition, fungal sporulation and bacterial growth were assessed. Fungal assemblages consisting of Articulospora tetracladia, Neonectria lugdunensis, Tricladium splendens and Varicosporium elodeae were used to study litter decomposition in microcosms exposed to NPs for 21 days. Two freshwater bacterial species belonging to the family Enterobacteriaceae were used to assess growth after 12 h of exposure to NPs. Types and concentrations of NPs affected litter decomposition and sporulation but not growth of bacteria. Leaf mass loss was significantly different between all concentrations but not between 25 and 50 µM. Fungal sporulation was significantly different among all concentrations of NPs. Fungal sporulation decreased with increase in concentration of NPs whereas a particular trend was not observed with mass loss except for Ag/ZnO. A 50 µM of Ag/ZnO had the highest impact on litter decomposition while 50 µM of ZnO on sporulation. The impact of Ag loaded metal oxides were not strikingly different from those of bare metal oxides except for the more pronounced impact on the mass loss accomplished by Ag/ZnO than by ZnO. Overall our study highlights that very low concentrations of NPs in freshwaters can impact freshwater ecosystem functioning.

Biodiversity of leaf litter fungi in streams along a latitudinal gradient.

Global patterns of biodiversity have emerged for soil microorganisms, plants and animals, and the extraordinary significance of microbial functions in ecosystems is also well established. Virtually unknown, however, are large-scale patterns of microbial diversity in freshwaters, although these aquatic ecosystems are hotspots of biodiversity and biogeochemical processes. Here we report on the first large-scale study of biodiversity of leaf-litter fungi in streams along a latitudinal gradient unravelled by Illumina sequencing. The study is based on fungal communities colonizing standardized plant litter in 19 globally distributed stream locations between 69°N and 44°S. Fungal richness suggests a hump-shaped distribution along the latitudinal gradient. Strikingly, community composition of fungi was more clearly related to thermal preferences than to biogeography. Our results suggest that identifying differences in key environmental drivers, such as temperature, among taxa and ecosystem types is critical to unravel the global patterns of aquatic fungal diversity.

Articulospora - Phylogeny vs morphology.

The taxonomy of the aquatic hyphomycete genus Articulospora (Ascomycota, Pezizomycotima, Leotiales, Helotiaceae) is based on the morphology of the generative phase of its lifecycle. The type species is Articulospora tetracladia, which is distributed worldwide. Its most frequent populations in nature have dimorphic conidia, differing by the extent of conidial branching (i.e., one or two levels of branching). Some strains, stable in culture, produce exclusively conidia of one type. With the molecular analyses employed here and the relatively low number of available isolates (20), separation based on branching of conidia has not been fully supported. Therefore we propose to retain the broad concept of A. tetracladia with dimorphic conidia. Among the three gene sequences tested as potential barcodes, the internal transcribed spacer (ITS) gene was the most promising region. All strains yielded amplifiable DNA which provided adequate resolution, according to accepted ranges in inter/intraspecific differences, to differentiate among the three Articulospora and two Fontanospora species that were tested (Articulospora atra, Articulospora proliferata, A. tetracladia, Fontanospora eccentrica, Fontanospora fusiramosa). D1/D2 primers also permitted amplification in all strains, however without much resolution. Amplification of the COX1 gene sequence was least consistent.

Does the developmental stage and composition of riparian forest stand affect ecosystem functioning in streams?

A common watershed restoration practice to improve water quality and stream ecosystem functions and services is replanting riparian corridors with plant species that may differ from those of natural communities. This restoration practice may have consequences on the aquatic ecosystem processes because organisms obtain energy from leaf litter inputs of the riparian zones. Leaf litter decomposition in streams is a vital ecosystem-level process, which depends on the activity of microorganisms and invertebrates. In the current study, we examined whether the type and developmental stage of riparian forest affect stream ecosystem functioning. We selected three widespread tree species in the Northwest Portugal, namely alder (Alnus glutinosa (L.) Gaertn.), oak (Quercus robur L.) and eucalyptus (Eucalyptus globulus Labill.) and conducted stream litter decomposition experiments with leaf litter from trees differing in developmental stage to assess leaf mass loss, fungal and invertebrate biomass and diversity. Both type and developmental stage of riparian stand significantly affected leaf mass loss, biomass of fungi and benthic invertebrates, sporulation of fungi, and abundance of invertebrates. However, only developmental stage of the riparian stand had an impact on the richness and diversity of fungi, whereas invertebrate diversity and richness was influenced by both stage and type classes. Overall our study provides the novel information that stream ecosystem processes are dictated not only by the composition but also by the developmental stage of the riparian stand. Moreover, this study provides an insight into how by altering riparian forest community composition through restoration practices may have an impact on a key ecosystem process and may have implications for successfully implementing future management strategies.

Copper tolerant ecotypes of Heliscus lugdunensis differ in their ecological function and growth.

Metal tolerance in aquatic hyphomycetes varies with the level of pollution at the fungal isolation site. While the focus of previous research has been on the effects of metal exposure on interspecies diversity, intraspecies variation of aquatic hyphomycetes remains largely unexplored. In this study we investigate the effects of Cu on ecological function (litter decomposition) and growth of five strains of Heliscus lugdunensis, isolated from contaminated and un-contaminated streams, in order to examine whether strains are expressed as ecotypes with distinct growth and functional signatures in response to metal stress. When exposed to Cu, strains of H. lugdunensis differed significantly in their litter decomposition and reproductive activity (sporulation) as well as mycelial growth, corresponding to the Cu concentrations at their isolation site. Strains isolated from sites with high Cu concentrations induced the highest litter decomposition or invested most in growth. This study broadens our understanding of Cu pollution in streams, which may lead to evolved adaptations of Cu tolerant ecotypes of H. lugdunensis differing in their ecological function, behaviour and morphology when exposed to metals.

Natural organic matter alters size-dependent effects of nanoCuO on the feeding behaviour of freshwater invertebrate shredders.

Nanoparticle size and the presence of natural organic matter (NOM) may influence the toxicity of nanoCuO to aquatic biota, but their interactive effects have been poorly investigated. We examined the feeding behaviour of the invertebrate shredder Allogamus ligonifer when exposed to sublethal concentrations of nanoCuO (50 and 100 mg L(-1)) with three particle sizes (12, 50 and 80 nm) in the absence or presence of humic acid (HA, 100 mg L(-1)) as a proxy of NOM. We further examined the ability of invertebrates to recover from the stressors. In the absence of nanoCuO and HA, the feeding rate of shredders was 0.416 mg leaf DM mg(-1 )animal DM day(-1). The exposure to increased nanoCuO concentrations inhibited the feeding rate and effects were stronger as nanoparticle size decreased (up to 83.3% inhibition for 12 nm particles). The exposure to HA alone inhibited the feeding activity by 52.7%. However, the co-exposure to nanoCuO and HA alleviated the inhibitory effects promoted by smaller and medium sized nanoCuO (up to 29.5%). The recovery of feeding activity by the shredders after stress removal was very low; maximum recovery (16.7%) was found for invertebrates rescued from pre-exposure to lower concentration of nanoCuO with larger size.

Fungi from metal-polluted streams may have high ability to cope with the oxidative stress induced by copper oxide nanoparticles.

Increased commercialization of products based on metal oxide nanoparticles increases the likelihood that these nanoparticles will be released into aquatic environments, thus making relevant the assessment of their potential impacts on aquatic biota. Aquatic fungi are distributed worldwide and play a key role in organic matter turnover in freshwater ecosystems. The present study investigated the impacts of copper oxide spherical nanoparticles (CuO-NPs; <50 nm powder, 5 levels ≤200 mg/L) on cellular targets and antioxidant defenses in 5 fungal isolates collected from metal-polluted or nonpolluted streams. The CuO-NPs induced oxidative stress in aquatic fungi, as evidenced by intracellular accumulation of reactive oxygen species, and led to plasma membrane damage and DNA strand breaks in a concentration-dependent manner. Effects were more pronounced with a longer exposure time (3 d vs 10 d). Under CuO-NP exposure, mycelia of fungi collected from metal-polluted streams showed less oxidative stress and higher activities of superoxide dismutase and glutathione reductase compared with fungi from nonpolluted streams. The latter fungi responded to CuO-NPs with a stronger stimulation of glutathione peroxidase activity. These findings may indicate that fungi isolated from metal-polluted streams had a greater ability to maintain the pool of reduced glutathione than those from nonpolluted streams. Overall, results suggest that populations adapted to metals may develop mechanisms to cope with the oxidative stress induced by metal nanoparticles.

Polyhydroxyfullerene binds cadmium ions and alleviates metal-induced oxidative stress in Saccharomyces cerevisiae.

The water-soluble polyhydroxyfullerene (PHF) is a functionalized carbon nanomaterial with several industrial and commercial applications. There have been controversial reports on the toxicity and/or antioxidant properties of fullerenes and their derivatives. Conversely, metals have been recognized as toxic mainly due to their ability to induce oxidative stress in living organisms. We investigated the interactive effects of PHF and cadmium ions (Cd) on the model yeast Saccharomyces cerevisiae by exposing cells to Cd (≤5 mg liter(-1)) in the absence or presence of PHF (≤500 mg liter(-1)) at different pHs (5.8 to 6.8). In the absence of Cd, PHF stimulated yeast growth up to 10.4%. Cd inhibited growth up to 79.7%, induced intracellular accumulation of reactive oxygen species (ROS), and promoted plasma membrane disruption in a dose- and pH-dependent manner. The negative effects of Cd on growth were attenuated by the presence of PHF, and maximum growth recovery (53.8%) was obtained at the highest PHF concentration and pH. The coexposure to Cd and PHF decreased ROS accumulation up to 36.7% and membrane disruption up to 30.7% in a dose- and pH-dependent manner. Two mechanisms helped to explain the role of PHF in alleviating Cd toxicity to yeasts: PHF decreased Cd-induced oxidative stress and bound significant amounts of Cd in the extracellular medium, reducing its bioavailability to the cells.

Physiological responses to nanoCuO in fungi from non-polluted and metal-polluted streams.

Nanocopper oxide (nanoCuO) is among the most widely used metal oxide nanoparticles which increases their chance of being released into freshwaters. Fungi are the major microbial decomposers of plant litter in streams. Fungal laccases are multicopper oxidase enzymes that are involved in the degradation of lignin and various xenobiotic compounds. We investigated the effects of nanoCuO (5 levels, ≤ 200 mg L(-1)) on four fungal isolates collected from metal-polluted and non-polluted streams by analyzing biomass production, changes in mycelial morphology, laccase activity, and quantifying copper adsorbed to mycelia, and ionic and nanoparticulate copper in the growth media. The exposure to nanoCuO decreased the biomass produced by all fungi in a concentration- and time-dependent manner. Inhibition of biomass production was stronger in fungi from non-polluted (EC50(10 days) ≤ 31 mg L(-1)) than from metal-polluted streams (EC50(10 days) ≥ 65.2 mg L(-1)). NanoCuO exposure led to cell shrinkage and mycelial degeneration, particularly in fungi collected from non-polluted streams. Adsorption of nanoCuO to fungal mycelia increased with the concentration of nanoCuO in the medium and was higher in fungi from non-polluted streams. Extracellular laccase activity was induced by nanoCuO in two fungal isolates in a concentration-dependent manner, and was highly correlated with adsorbed Cu and/or ionic Cu released by dissolution from nanoCuO. Putative laccase gene fragments were also detected in these fungi. Lack of substantial laccase activity in the other fungal isolates was corroborated by the absence of laccase-like gene fragments.

Preliminary insights into the phylogeography of six aquatic hyphomycete species.

Aquatic hyphomycetes occur worldwide on a wide range of plant substrates decomposing in freshwaters, and are known to play a key role in organic matter turnover. The presumed worldwide distribution of many aquatic hyphomycete species has been based on morphology-based taxonomy and identification, which may overlook cryptic species, and mask global-scale biogeographical patterns. This might be circumvented by using DNA sequence data. The internal transcribed spacer (ITS) region from rDNA was recently designated as the most suitable barcode for fungal identification. In this study, we generated ITS barcodes of 130 isolates belonging to 6 aquatic hyphomycete species (Anguillospora filiformis, Flagellospora penicillioides, Geniculospora grandis, Lunulospora curvula, Tetrachaetum elegans and Tricladium chaetocladium), and collected from streams of Southwest Europe (86 isolates) and East Australia (44 isolates). European and Australian populations of 4 species (A. filiformis, F. penicillioides, G. grandis and T. elegans) grouped into different clades, and molecular diversity indices supported significant differentiation. Continents did not share haplotypes, except for T. chaetocladium. Overall results show substantial population diversity for all tested species and suggests that the biogeography of aquatic hyphomycetes may be species-specific.

Copper oxide nanoparticles can induce toxicity to the freshwater shredder Allogamus ligonifer.

Increased commercialisation of nanometal-based products augments the possibility of their deposition into aquatic ecosystems; this, in turn, may pose risks to aquatic biota and associated ecological functions. Freshwater invertebrate shredders mostly use microbially-colonized plant litter as food resource and play an important role in aquatic detritus food webs. We assessed lethal effects of nanoCuO on the shredder Allogamus ligonifer (Trichoptera, Limnephilidae) by determining the concentration that induced 50% of death (LC(50)), and sublethal effects of nanoCuO on the feeding behaviour and growth of the shredder by exposing the animals to: (i) stream water supplemented with nanoCuO and microbially-colonized leaves, and (ii) stream water (without nanoCuO) and microbially-colonized leaves pre-exposed to nanoCuO. Results from acute lethal tests showed that the 96 h LC(50) of nanoCuO was very high (569 mg L(-1)). In the absence of nanoparticles, leaf consumption rate was 0.27 mg leaf DM mg(-1) animal DM d(-1) and the shredder growth rate was 56 µg animal DM mg(-1) animal DM d(-1). A significant inhibition in leaf consumption rate (up to 47%) and invertebrate growth rate (up to 46%) was observed when shredders were exposed to the higher tested sublethal concentration of nanoCuO (75 mg L(-1)) through either contaminated stream water or pre-contaminated food. The exposure to increased nanoCuO concentration via water or pre-contaminated food led to higher accumulation of copper in the larval body. Leached water-soluble ionic copper from the nanoCuO adsorbed or accumulated in the shredder (up to 10.2% of total Cu) seemed to influence the feeding behaviour and growth of the shredder.

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.