Antioxidative responses of duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis> L.) to short-term copper exposure

Goal, Scope and Background

Elevated concentrations of copper in the environment result in accumulation of the metal in plants and cause an increase in reactive oxidative species (ROS). The first response to elevated amounts of ROS is increased levels of enzymatic and non-enzymatic antioxidants that reduce oxidative stress. The aim of our study was to evaluate the early stages of antioxidative responses to the low copper concentrations usually present in moderately polluted environments. In addition, some other parameters were examined to evaluate the effect of copper on plants.

Methods

Duckweed (Lemna minor L.) was exposed to different concentrations of copper sulphate for up to 24 hours. Glutathione concentration and enzymatic activities of catalase, guaiacol peroxidase and glutathione reductase were measured spectrophotometrically. Additionally, delayed and prompt chlorophyll fluorescence was measured by luminometry and fluorometry, respectively. The accumulation of copper in plants exposed for 24 hours to various concentrations of copper sulphate was measured by flame atomic absorption spectrophotometry.

Results

The treatment of plants with copper sulphate resulted in an immediate decrease of the glutathione pool, which was replenished after 24 hours at CuSO₄ concentrations lower than 2 μM. Higher CuSO₄ concentrations caused a decrease of reduced glutathione. The responses of the antioxidant enzymes glutathione reductase, guaiacol peroxidase and catalase to CuSO₄ differed during the first six hours of exposure, but their enzyme activities all increased after 24 hours of exposure. All these enzymes displayed biphasic activity curves with maximum values between 0.5 μM and 1 μM CuSO₄. The response of guaiacol peroxidase was the most pronounced and statistically significantly specific and that of catalase the least. Delayed chlorophyll fluorescence decreased after exposure to 1 μM CuSO₄, but no significant effect on maximum quantum yield of photosystem II (Fv/Fm) was observed. L. minor accumulated relatively high concentrations of copper. The accumulation rate was higher at lower concentrations of copper in the test medium (up to 2 μM CuSO₄) than at concentrations above 2 μM CuSO₄.

Discussion

One of the most pronounced antioxidative responses to copper exposure was modified levels of oxidized and reduced forms of glutathione. The decrease of the glutathione pool is most probably coupled with induced production of phytochelatins. Antioxidative enzymes showed the biphasic enzyme activity characteristic of stress response. Guaiacol peroxidase exhibited the greatest significant increase of activity, even at higher CuSO₄ concentrations at which the activity of catalase and glutathione reductase dropped. The intensity of delayed chlorophyll fluorescence decreased, indicating reduced photosynthesis of plants under stress. All the measured parameters showed that plants respond to even low copper concentrations very soon after exposure. The accumulation rate of copper in duckweed tissues indicates that L. minor is an accumulator species.

Conclusions

The synchronized and prompt inducibility of antioxidants indicates their involvement in a general plant defence strategy for coping with metal-induced oxidative stress. Glutathione concentration and guaiacol peroxidase activity were found to be the most sensitive of the early indicators of exposure to copper concentrations present in polluted water bodies.

Recommendation and Perspectives

The experimental design of the present study allowed us to compare the sensitivity of various methods and parameters for detecting plant responses to heavy metal-induced oxidative stress. The level of glutathione and the enzyme activities of guaiacol peroxidase and glutathione reductase could be used as a rapidly determined early warning system in toxicity studies.

     

Age dependent acute oral toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and two anaerobic N-nitroso metabolites in deer mice (Peromyscus maniculatus)

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transforms anaerobically into N-nitroso compounds: hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Exposure to these N-nitroso metabolites may occur in areas contaminated with explosives, as anaerobic degradation occurs via some bacteria and is one remediation strategy used for RDX. Few papers report acute oral toxicity and none have evaluated age dependent toxicity of RDX or its N-nitroso metabolites. Median lethal dose (LD₅₀) was determined in deer mice (Peromyscus maniculatus) of three age classifications 21 d, 50 d, and 200 d for RDX, MNX, and TNX using the US EPA up-and-down procedure (UDP). Hexahydro-1,3,5-trinitro-1,3,5-triazine and N-nitroso metabolites caused similar overt signs of toxicity. Median lethal dose for 21 d deer mice were 136, 181, and 338 mg/kg for RDX, MNX, and TNX, respectively. Median lethal dose for 50 d deer mice were 319, 575, and 338 mg/kg for RDX, MNX, and TNX, respectively. Median lethal dose for 200 d deer mice were 158, 542, and 999 mg/kg for RDX, MNX, and TNX, respectively. These data suggest that RDX is the most potent compound tested, and age dependent toxicity may exist for all compounds and could play a role in RDX and RDX N-nitroso metabolite ecological risk evaluation of terrestrial wildlife at RDX contaminated sites.     

More than euglossines: the diverse pollinators and floral scents of Zygopetalinae orchids

Floral volatile organic compounds (VOCs) play important roles in plant-pollinator interactions. We investigated the reproductive ecology and floral VOCs of Zygopetalinae orchids to understand the relationship between floral scents and pollinators. We performed focal observations, phenological censuses and breeding system experiments in eight species in southeast Brazil. Floral scents were collected and analysed using SPME/GC-MS. We performed multivariate analyses to group species according to affinities of their VOCs and define compounds associated to each plant. Dichaea cogniauxiana was pollinated by weevils which use their developing ovules, while D. pendula was pollinated by the same weevils and perfume-collecting male euglossine bees. The other species were deceit-pollinated by bees. Zygopetalum crinitum was pollinated by carpenter bees, while W. warreana, Z. mackayi and Z. maxillare were bumblebee-pollinated. The latter was also pollinated by Centris confusa. Breeding system varied widely with no association to any pollinator group. Most VOCs are common to other floral scents. Zygopetalum crinitum presented an exclusive blend of VOCs, mainly composed of benzenoids. The scents of Pabstia jugosa, Promenaea xanthina and the Zygopetalum spp. were similar. The bumblebee-pollinated species have flowering periods partially overlapped, thus neither phenology nor pollinators constitute hybridization barriers among these species. Euglossines are not the only pollinators of Zygopetalinae. Different VOCs, size and lifespan of flowers are associated with distinct pollinators. A distinctive VOC bouquet may determine specialisation in carpenter bees or male euglossines within bee-pollinated flowers. Finally, visitation of deceit-pollinated flowers by perfume-collecting euglossines allows us to hypothesise how pollination by this group of bees had evolved.     

Detection of Torque Teno Sus Virus in Pork Bile and Liver Sausages

Torque teno viruses (TTV) are small DNA viruses widespread among humans and pigs. The clinical significance of TTV infections in either humans or pigs is uncertain. In fact, TTV viremia is highly prevalent in patients with different pathologies, but it can also be frequently observed in healthy subjects. Virus infection in pigs is considered a putative cofactor in several diseases; despite being detected frequently in healthy animals, its role still remains unknown. The present study aimed to investigate the presence of Torque teno sus virus (TTSuV) in 62 bile samples collected from pigs at slaughterhouse and in 36 fresh pork liver sausages bought at point of sale. Quantitative Real-Time PCR, confirmed that 19.4 and 58.3 % of bile and sausage samples tested positive for TTSuV, respectively. The mean viral load was established as 5.6 × 10⁴ GE/µl for bile and 7.16 × 10³ GE/g for sausages. TTSuV nucleotide sequence analysis confirmed a wide heterogeneity among the circulating TTSuV strains, which included both TTSuV1 and TTSuV2.     

Comparison of the Activity of Alcohol-Based Handrubs Against Human Noroviruses Using the Fingerpad Method and Quantitative Real-Time PCR

Noroviruses (NoV) are the most common cause of acute nonbacterial gastroenteritis in the United States, and human hands play an important role in their transmission. Little is known about the efficacy of hand hygiene agents against these highly infectious pathogens. We investigated the activity of seven commercially available hand hygiene products against human noroviruses by in vivo fingerpad tests. The in vivo activity of alcohol-based handrubs ranged from 0.10 to 3.74 log reduction and was not solely dependent on alcohol concentration. A handrub (VF481) based on 70% ethanol and a blend of other skin care ingredients reduced Norwalk virus (NV) by 3.74 log in 15 s and provided significantly greater NV reduction than all the other products tested (P < 0.001). Furthermore, VF481 was the most effective product tested against the NoV genogroup II strains Snow Mountain virus (GII.2) and a GII.4 strain. These results demonstrate that alcohol by itself is not effective against NoV, but effective formulation of alcohol-based handrubs can achieve significant reduction of norovirus RNA on fingers.     

Modelling spatial patterns of correlations between concentrations of heavy metals in mosses and atmospheric deposition in 2010 across Europe

Background

This paper aims to investigate the correlations between the concentrations of nine heavy metals in moss and atmospheric deposition within ecological land classes covering Europe. Additionally, it is examined to what extent the statistical relations are affected by the land use around the moss sampling sites. Based on moss data collected in 2010/2011 throughout Europe and data on total atmospheric deposition modelled by two chemical transport models (EMEP MSC-E, LOTOS-EUROS), correlation coefficients between concentrations of heavy metals in moss and in modelled atmospheric deposition were specified for spatial subsamples defined by ecological land classes of Europe (ELCE) as a spatial reference system. Linear discriminant analysis (LDA) and logistic regression (LR) were then used to separate moss sampling sites regarding their contribution to the strength of correlation considering the areal percentage of urban, agricultural and forestry land use around the sampling location. After verification LDA models by LR, LDA models were used to transform spatial information on the land use to maps of potential correlation levels, applicable for future network planning in the European Moss Survey.

Results

Correlations between concentrations of heavy metals in moss and in modelled atmospheric deposition were found to be specific for elements and ELCE units. Land use around the sampling sites mainly influences the correlation level. Small radiuses around the sampling sites examined (5 km) are more relevant for Cd, Cu, Ni, and Zn, while the areal percentage of urban and agricultural land use within large radiuses (75–100 km) is more relevant for As, Cr, Hg, Pb, and V. Most valid LDA models pattern with error rates of <?40% were found for As, Cr, Cu, Hg, Pb, and V. Land use-dependent predictions of spatial patterns split up Europe into investigation areas revealing potentially high (=?above-average) or low (=?below-average) correlation coefficients.

Conclusions

LDA is an eligible method identifying and ranking boundary conditions of correlations between atmospheric deposition and respective concentrations of heavy metals in moss and related mapping considering the influence of the land use around moss sampling sites.

     

Hazardous and essential trace elements profile in the different soft tissues of Lithophaga lithophaga (Linnaeus, 1758) from Southern Adriatic Sea (Italy)

A characterization study of 7 hazardous (Hg, Pb, Cd, As, Cr, U, and Ni) and 11 essential (Co, Mo, Se, Cu, Zn, V, Ca, Al, Sr, Mn, and Fe) trace elements in date mussels (Lithophaga lithophaga,Linnaeus, 1758) was carried out by inductively coupled plasma mass spectrometer. Date mussels (231 individuals), caught in the Gulf of Manfredonia (Southern Italy), were divided into 4 size-related groups. The different tissues (muscle, stomach, hepatopancreas and rest of soft tissues) were separated and analyzed to study the capability of absorption of this species. No particular differences were reported on the basis of the shell size; the soft tissues play an important role on the accumulation levels of hazardous and essential trace elements. These marine animals may be considered a good bioindicator of marine environmental pollution for their longevity and habitat permanence peculiarities.     

Assessment of Atmospheric Deposition of Heavy Metals and Other Elements in Belgrade Using the Moss Biomonitoring Technique and Neutron Activation Analysis

This study aimed at assessing atmospheric deposition of heavy metals and other elements using the moss genera Brachythecium sp. (B. rutabulum and B. salebrosum) and Eurhynchium sp. (E. hians and E. striatum) collected in autumn 2004 in the urban area of Belgrade. The concentrations of 36 elements (Na, Mg, Al, Cl, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Zn, As, Se, Br, Rb, Sr, Mo, Sb, I, Cs, Ba, La, Ce, Sm, Tb, Dy, Hf, Ta, W, Hg, Th, U) were determined in moss and local topsoil samples by instrumental neutron activation analysis. The concentration of elements in moss positively correlated to those obtained for topsoil. High enrichment factors for As, Zn, Mo, Br, Sb, Se, Hg and Cl, calculated to continental crust composition, gave an evidence for anthropogenic impact on urban area, mainly due to intensive vehicular traffic and fossil fuel combustion. The concentration of elements in moss, characteristic for fossil fuel combustion, obtained in this study were substantially lower than in the previous investigation (2000) conducted in the area of Belgrade. The level of concentrations for V, Cr, Ni, and As in moss from this study correlated to those measured for neighboring countries, and were several times higher than the base-level data from low polluted areas. The level of accumulated elements in both investigated moss genera were similar and all studied species could be combined for biomonitoring purposes in urban areas.     

Elemental Composition of Urban Aerosol Collected in Florence, Italy

An extensive investigation is in progress aiming at the characterisation of the air particulate composition in Florence. For our investigation, we use the external PIXE-PIGE beam facility of the I.N.F.N. Van de Graaff accelerator at the Physics Department of the Florence University. In order to gather information on both the longer- and shorter-time trends of the aerosol elemental composition, we are analysing both long temporal series (about 1 year) of 24-h Millipore filters collected by the health authorities in 3 different sites, and filters collected simultaneously in two of the above sites for about one month, by two streakers with one-hour resolution, providing size fractionation between particle size smaller than 2.5 µm and from 2.5 µm to 10 µm. The streakers sampling period includes two days during which the Municipality of Florence has banned the circulation of non-catalytic cars, due to the increase of NO₂ above the "recommended safety values". We present here the first obtained results for the sampling site located near a heavy traffic road.     

The early bee catches the flower - circadian rhythmicity influences learning performance in honey bees, <Emphasis Type="Italic">Apis mellifera</Emphasis>

Circadian rhythmicity plays an important role for many aspects of honey bees’ lives. However, the question whether it also affects learning and memory remained unanswered. To address this question, we studied the effect of circadian timing on olfactory learning and memory in honey bees Apis mellifera using the olfactory conditioning of the proboscis extension reflex paradigm. Bees were differentially conditioned to odours and tested for their odour learning at four different “Zeitgeber” time points. We show that learning behaviour is influenced by circadian timing. Honey bees perform best in the morning compared to the other times of day. Additionally, we found influences of the light condition bees were trained at on the olfactory learning. This circadian-mediated learning is independent from feeding times bees were entrained to, indicating an inherited and not acquired mechanism. We hypothesise that a co-evolutionary mechanism between the honey bee as a pollinator and plants might be the driving force for the evolution of the time-dependent learning abilities of bees.