Multiple responses of Mytilus galloprovincialis to plastic microfibers.

Plastic microfibers (MF) represent the major source of MF found in the environment, the majority made of polyester (PES). Marine bivalves, suspension feeders widespread in coastal areas subjected to higher anthropogenic input, can accumulate MF from the water column in their tissues. This raised some concern about their possible impact on bivalve health and potential transfer along the food chain. In this work, the effects of PES-MF on the mussel Mytilus galloprovincialis were investigated, utilizing MF obtained by cryo-milling of a fleece cover. Fiber characterization indicated the polymer composition as polyethylene terephthalate (PET); the size distribution was in a length range resembling that of MF released from textile washing, and including those that can be ingested by mussels. MF were first screened for short-term in vitro immune responses in mussel hemocytes. The effects of in vivo exposure (96 h, 10 and 100 µg/L, corresponding to about 150 and 1500 MF/mussel/L, respectively), were then evaluated. Data are presented on hemolymph immune biomarkers (Reactive Oxygen Species and nitric oxide production, lysozyme activity), and on antioxidant biomarkers (catalase and glutathione S-transferase) and histopathology in gills and digestive gland. Tissue MF accumulation was also evaluated. MF exposure stimulated extracellular immune responses both in vitro and in vivo, indicating induction of immune/inflammatory processes. In both tissues, stimulation of antioxidant enzyme activities, suggesting oxidative stress conditions, and histopathological changes were observed, with stronger effects often observed at lower concentration. Although mussel retained a very small fraction of MF, their accumulation was higher in the digestive gland than in gills, and in both tissues of mussels exposed to the lowest concentration. Selective accumulation of shorter MF was also observed, particularly in gills. Overall, the results demonstrate that at environmental exposure levels, PET-MF have a significant impact on mussel physiology, affecting multiple processes in different tissues.

Proteomic analyses of early response of unicellular eukaryotic microorganism Tetrahymena thermophila exposed to TiO2 particles.

Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO2 nanoparticles (TiO2-NPs) and their bulk counterparts (bulk-TiO2). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO2-NPs or bulk-TiO2 particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO2 particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO2-NPs concentration differed significantly from the response to higher TiO2-NPs concentration and both bulk-TiO2 concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO2; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO2-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.

Acclimation of Tetrahymena thermophila to bulk and nano-TiO2 particles by changes in membrane fatty acids saturation.

We provide experimental evidence that changes in the membrane fatty acid profile of Tetrahymena thermophila incubated with nano- or bulk TiO(2) particle are not accompanied by ROS generation or lipid peroxidation. Consequently these changes are interpreted as acclimation to unfavorable conditions and not as toxic effects. T. thermophila cells were exposed to TiO(2) particles at different concentrations for 24h at 32°C. Treatment of cultures with nano- and bulk TiO(2) particles resulted in changes of membrane fatty acid profile, indicating increased membrane rigidity, but no lipid peroxidation or ROS generation was detected. There were no differences in membrane composition when T. thermophila was exposed to nanosized or bulk-TiO(2) particles. We also observed reversible filling of food vacuoles, but this was different in case of nano- or bulk TiO(2) exposure. Our results suggest that interactions of particles and cell membranes are independent of oxidative stress.

Use of a modified Allium test with nanoTiO2.

Extensive production and wide application of TiO(2) nanoparticles has stimulated research on its potential biological effects on different groups of organisms but the interaction of TiO(2) nanoparticles with higher plants remains poorly understood. We have studied the effect of TiO(2) nanoparticles on Allium cepa using a modification of the conventional Allium test with nanoparticles suspended in distilled water as opposed to growth medium. Nanoparticulate TiO(2) was found to have low toxic potential and the mitotic index was among the most sensitive measures of the effect of nano-TiO(2). We conclude that modified Allium test is suitable to provide comparative data on the biological potential of a variety of nanoparticles and could be used in a tiered approach to nanotoxicity testing.

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation.

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.

Long-term Hg pollution induced Hg tolerance in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea).

The aim of our work was to assess the pollution-induced community tolerance (PICT) of isopod gut microbiota and pollution-induced isopod population tolerance (PIPT). Animals collected from a chronically Hg polluted and an unpolluted location were exposed for 14 days to 10microg Hg/g dry food under laboratory conditions. The lysosomal membrane stability, hepatopancreas epithelium thickness, feeding activity and animal bacterial gut microbiota composition were determined. The results confirm the hypothesis that the response to short-term Hg exposure differs for animals from the Hg polluted and the unpolluted field locations. The animals and their gut microbiota from the Hg polluted location were less affected by Hg in a short-term feeding experiment than those from the unpolluted environment. We discuss the pollution-induced population tolerance of isopods and their gut microbiota as a measure of effects of long-term environmental pollution. The ecological consequences of such phenomena are also discussed.

Focused ion beam characterization of plasma-assisted deposition on polymer films at the nanoscale.

In this paper, a novel technique is presented for the characterization at the nanoscale of plasma-assisted deposit on polyethylene-terephthalate (PET) polymer films. In previous studies, some microcharacterization and morphology analyses of plasma-assisted deposition were performed by atomic force microscopy (AFM). In the work presented here, we analysed the thickness and homogeneity of plasma-assisted deposits by focused ion beam (FIB). This technique with 5-7 nm resolution requires no sample preparation and relies on a sequence of operations on a relatively fast time scale, so that it is easy to make thorough investigations of the sample. We performed electron and ion imaging of the surface of the material, and a subsequent ionic cutting allowed the study of the morphology of the same sample. We developed a novel approach to the edge detection techniques (EDT) in images for a fast evaluation and monitoring of the deposited layer.

Electron and ion imaging of gland cells using the FIB/SEM system.

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga(+) ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga(+) ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.

Anaerobic bacteria in the gut of terrestrial isopod Crustacean Porcellio scaber.

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.

Morphological description of bacterial infection of digestive glands in the terrestrial isopod porcellio scaber (Isopoda, crustacea)

Morphological studies of the hepatopancreas of the terrestrial isopod Porcellio scaber revealed bacterial infection. The percentage of infected animals collected from the same site varied from 0 to 10% during the 4 years of study. Transmission electron microscopy, scanning electron microscopy, and light microscopy revealed that infected glands differed from those in healthy isopods. The most prominent sign was white spots between 100 and 200 &mgr;m in diameter along the entire gland. These spots were aggregations of vacuoles in the cells that were densely filled with bacteria in different phases of the developmental cycle that included the formation of small, dense, rod-shaped infective bacteria and much larger spherical multiplying cells filled with aggregates of polysomes and a chromatin network. Occasionally, large sphericles were filled with homogeneous electron-dense material. Bacteria were not observed in the cell nucleus. Small vacuoles of less than 5 &mgr;m were filled predominately with spherical bacteria but rod-shaped forms were also present in large numbers. Larger vacuoles of 10 to 20 &mgr;m in the main were densely filled with rod-shaped bacteria. According to the literature on the morphological characteristics of bacteria infecting invertebrates, those described in our study would be classified in the genus Rickettsiella. However, most recent investigations show that besides morphological investigations, genetic ones are also needed to define the taxonomic position of bacteria that infect invertebrates. Copyright 1999 Academic Press.

The effect of Zn on the digestive gland epithelium of Porcellio scaber (Isopoda, Crustacea).

The effect of zinc (Zn)-contaminated food on the shape of digestive gland epithelium was studied in the terrestrial isopod, Porcellio scaber. In animals fed with 5,000 micrograms Zn g-1 dry wt. of food, the epithelium was flattened in the anterior part of the gland tube. In the posterior part of the gland tube, the shape and size of cells did not change drastically, but they had folded apical surface. In animals fed with more Zn-contaminated food (10,000 micrograms Zn g-1 dry wt.) the epithelium was uniformly flattened and the basal lamina was intensively folded. In both cases, reduction of lipid bodies was evident. The possibility of using the shape of the gland epithelium as a biomarker of toxic chemicals is discussed.

The toxicity of zinc to terrestrial isopods in a "standard" laboratory test.

A method is described for assessing the effects of metals on the food consumption rate of isopods from measurements of fecal production. The effects of zinc in the diets of two isopod species, Porcellio scaber and Oniscus asellus, were tested. The metal was fed to the isopods on leaves of field maple (Acer campestre) contaminated with concentrations ranging between 1000 and 10,000 micrograms Zn g-1 leaf dry wt. Significant reductions in feeding rates were observed of the highest concentrations of zinc. The test employed in this study is quick, cheap, and relevant for estimating sublethal effects of metals on isopods.

Bacteria adherent to the hindgut of terrestrial isopods.

The gut microflora of three terrestrial isopod species was investigated by means of scanning electron microscope. The gut cuticle of intermoult specimens of Ligidium hypnorum and Porcellio scaber was densely colonized with bacteria. Some moulting specimens of the same species were observed with the guts free of bacteria. In all investigated specimens of Hyloniscus riparius the guts were completely free of microflora. In this case our finding could not be interpreted in relation to the moulting stage of the animal.