Sahadevan Seena

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.

Potential use of barcoding to identify aquatic hyphomycetes

Tetracladium is a common aquatic hyphomycete genus, whose taxonomy has been based on the morphology and development of asexual spores. Ecological surveys have relied almost exclusively on spore morphology. Since selective pressures have resulted in convergent shapes, misidentifications are a concern. To supplement morphological information, we determined COX1, ITS and D1/D2 sequences as potential barcodes on 21 strains belonging to 7 described Tetracladium species and an unidentified strain. Attempts to amplify the IGS region were unsuccessful. The ratio of intraspecific to interspecific variability was optimal with ITS, which also provided the intuitively most acceptable cladogram. Typical conidia and their variability for the seven described species are illustrated. Internal node reliability depended less on total sequence length and more on the mixture of conserved and variable regions used to build cladograms. This finding can be exploited for quickly increasing phylogenetic accuracy without greatly increasing the amount of amplification and sequencing. The results have important implications for identifying freshwater hyphomycetes in the field but further work is required to establish if this method works with plant pathogens.

Nutritional quality evaluation of velvet bean seeds (Mucuna pruriens) exposed to gamma irradiation

Effects of gamma irradiation on Mucuna pruriens seeds at various doses (0, 2.5, 5, 7.5, 10, 15 and 30 kGy) on the proximate composition, mineral constituents, amino acids, fatty acids and functional properties were investigated. Gamma irradiation resulted in a significant increase of crude protein at all doses, while the crude lipid, crude fibre and ash showed a dose-dependent decrease. Raw Mucuna seeds were rich in minerals (potassium, phosphorus, calcium, magnesium, iron and selenium). Sodium, copper and manganese were significantly decreased on irradiation at all the doses, while magnesium and iron showed a significant decrease only above 10 kGy. The essential amino acids of raw and gamma-irradiated Mucuna seeds were comparable with the FAO/WHO recommended pattern. A significant increase of in vitro protein digestibility was seen in seeds irradiated at 30 kGy. High amounts of unsaturated fatty acids in Mucuna seeds decreased significantly after irradiation. However, linoleic acid was not present in raw seeds but detected after irradiation and it was elevated to high level at 30 kGy. Behenic acid, a major anti-nutritional factor, was reduced significantly on irradiation, indicating the positive effect of gamma irradiation on Mucuna seeds. Significant enhancement in the water absorption and oil absorption capacities, protein solubility, emulsion activity and improvement in the gelation capacity was recorded after irradiation. Results of the present investigation reveal that application of gamma irradiation does not affect the overall nutritional composition and can be used as an effective method of preservation of Mucuna seed and their products.

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.

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.

Nutrient composition and biological evaluation of an unconventional legume, Canavalia cathartica of mangroves.

Seeds of an unconventional legume, Canavalia cathartica from the mangroves of the southwest coast of India, were screened for proximate composition, minerals, protein fractions, amino acid profiles, fatty acids and some anti-nutritional factors. The seeds consisted of 31.2%, 1.86%, 61.4% and 1580 kJ crude proteins, crude lipid, crude carbohydrates and calories, respectively. The crude protein content of seeds was higher than common cereals, whole-wheat flour (8.55%), parboiled rice (7.7%) and egg (12.6%). Essential amino acids, threonine, cysteine + methionine, isoleucine, tyrosine + phenylalanine and lysine were higher than Food and Agricultural Organization of United Nations/World Health Organization patterns. On comparing the amino acid profile of mangrove and sand dune C. cathartica seeds, the latter were found to be superior (total amino acid content, 123.5% versus 60.8%), while the result was vice versa with respect to the polyunsaturated/saturated ratio (105.9 versus 2.52). The seeds possessed 1420 mg/100 g total phenolics and strong hemagglutination activity, while tannins and trypsin inhibition activity were absent. Physical, nutritional and anti-nutritional features of seeds of C. cathartica of the mangrove have been compared with those of C. cathartica of the sand dunes of the southwest coast of India. A significant difference (P < 0.05) was observed between the physical, proximal and mineral composition except for ash, magnesium and copper. The protein efficiency ratio, food efficiency ratio, net protein retention, protein retention efficiency, biological value, net protein utilization and true digestibility of C. cathartica seeds were significantly different (P < 0.05) from casein. In spite of the high protein content, animal-feeding trials indicated low nutritional quality. Nutritional, anti-nutritional factors and protein qualities of seeds C. cathartica have been discussed in comparison with other species of Canavalia.

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.

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

ABSTRACT 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.