SEM-EDS and X-ray micro computed tomography studies of skeletal surface pattern and body structure in the freshwater sponge Spongilla lacustris collected from Goczalkowice reservoir habit (Southern Poland).

INTRODUCTION: Freshwater sponges are common animals of most aquatic ecosystems. They feed by filtering small particles from the water, and so are thought to be sensitive indicators of pollution. Sponges are strongly associated with the abiotic environment and are therefore used as bioindicators for monitoring of water quality in water habitats. Among the freshwater sponges, Spongilla lacustris is one of the classic models used to study evolution, gene regulation, development, physiology and structural biology in animal water systems. It is also important in diagnostic of aquatic environments. The aim of this study was to characterize and visualize three-dimensional architecture of sponge body and measure skeleton elements of S. lacustris from Goczalkowice reservoir for identification purposes. MATERIAL AND METHODS: The scanning electron microscopy with an energy dispersive X-ray microanalysis (SEM- -EDS) and X-ray micro computed tomography (micro-CT) were used to provide non-invasive visualization of the three-dimensional architecture of Spongilla lacustris body. RESULTS: We showed that sponge skeleton was not homogeneous in composition and comprised several forms of skeleton organization. Ectosomal skeleton occurred as spicular brushes at apices of primary fibres with cementing spongin material. Choanosomal skeletal architecture was alveolate with pauci- to multispicular primary fibres connected by paucispicular transverse fibres, made by megascleres embedded in a scanty spongin matrix both in the choanosome and at the sponge surface. In contrast, microscleres were irregularly scattered in choanosome and skeletal surface. Furthermore, SEM-EDS studies showed that the distribution of silica in megascleres and microscleres was observed along the spicules and sponge surface areas. CONCLUSIONS: In conclusion, we showed that the combination of SEM-EDS and micro-CT microscopy techniques allowed obtaining a complete picture of the sponge spatial architecture.

Elemental distribution in reproductive and neural organs of the Epilachna nylanderi (Coleoptera: Coccinellidae), a phytophage of nickel hyperaccumulator Berkheya coddii (Asterales: Asteraceae) by micro-PIXE.

The phenomenon of metal hyperaccumulation by plants is often explained by a pathogen or herbivore defense hypothesis. However, some insects feeding on metal hyperaccumulating plants are adapted to the high level of metals in plant tissues. Former studies on species that feed on the leaves of Berkheya coddii Roessler 1958 (Asteraceae), a nickel-hyperaccumulating plant, demonstrated several protective mechanisms involved in internal distribution, immobilization, and elimination of Ni from the midgut and Malpighian tubules. These species are mainly coleopterans, including the lady beetle, Epilachna nylanderi (Mulsant 1850) (Coleoptera: Coccinellidae), collected from the ultramafic ecosystem near Barberton in South Africa. By performing particle-induced X-ray emission microanalysis elemental microanalysis (PIXE), this study examined whether Ni may be harmful to internal body systems that decide on insect reactivity (central nervous system [CNS]), their reproduction, and the relationships between Ni and other micronutrients. Data on elemental distribution of nine selected elements in target organs of E. nylanderi were compared with the existing data for other insect species adapted to the excess of metals. Micro-PIXE maps of seven regions of the CNS showed Ni mainly in the neural connectives, while cerebral ganglia were better protected. Concentrations of other bivalent metals were lower than those of Ni. Testis, compared with other reproductive organs, showed low amounts of Ni. Zn was effectively regulated at physiological dietary levels. In insects exposed to excess dietary Zn, it was also accumulated in the reproductive organs. Comparison of E. nylanderii with other insects that ingest hyperaccumulating plants, especially chrysomelid Chrysolina clathrata (Clark) (Coleoptera: Chrysomelidae), showed lower protection of the CNS and reproductive organs.

X-ray microtomography for imaging of developing spiders inside egg cocoons.

Embryogenesis is especially sensitive to external factors. The changes in its course are often used as biomarkers of environmental impact. Since spider embryogenesis takes place inside cocoons, it is crucial to find a reliable tool to analyze this developmental phase with no intrusion into the cocoons. The aim of this study was to verify the efficacy of X-ray microtomography for non-invasive analysis of embryonic morphology and egg quantity in the cocoons of Xerolycosa nemoralis and Agelena labyrinthica from polluted and reference sites. X-ray microtomography slice images as well as 3D images and animations obtained from digital visualization of those slides were used to study the morphology of embryos and egg arrangement in the cocoons. Any disorders in embryogenesis or malformation of embryos in relation to site of origin have not been found, but inside an egg cocoon of X. nemoralis from the polluted site embryos differing form each other by one developmental stage were identified. Egg calculation revealed a K- reproductive strategy of X. nemoralis from polluted sites. Finally, future prospects and benefits, and weaknessess of this method for the study of spider cocoons have presented.

The effects of cadmium or zinc multigenerational exposure on metal tolerance of Spodoptera exigua (Lepidoptera: Noctuidae).

The effects of ten generational zinc or cadmium pre-exposure on metal tolerance among beet armyworm Spodoptera exigua individuals were compared. These effects were assessed in animals from the 11th generation, reared on a diet either uncontaminated or contaminated with metal (cadmium or zinc). The survival rate of larvae and the degree of metal accumulation (in larvae, pupae and moths; among larval organs: gut and fat body) were analysed. Catalase, superoxide dismutase and glutathione transferase activity in larval organs of individuals subjected to different metal treatments were also measured. Animals transferred from control rearing to metals (cadmium or zinc) in the 11th generation, as well as those from multigenerational zinc treatment, but not from multigenerational cadmium treatment, had a significantly lower survival rate than control animals. Insects from the groups with the high metal treatment had high bioaccumulation factors (above 3.7 and 2.3 following cadmium and zinc, respectively). Cadmium (but not zinc) pre-exposure had a significant effect on metal accumulation in larvae. Multigenerational metal pre-exposure seemed to have mainly a negative effect on glutathione transferase activity in the gut of larvae from the 11th generation, in the case of the individuals exposed to metal other than that used in pre-exposure treatment or kept in control conditions. However, in the case of zinc pre-exposure, such effect was only apparent when zinc was replaced by cadmium. The long-term effect of cadmium on catalase activity in larvae was found.

Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid.

In the present study we describe the effect of chloronicotinoid pesticide (imidacloprid) on the digestive enzymes activity of the Cameraria ohridella larvae after lasting 1 year sublethal exposure to imidacloprid pesticide. Caterpillars - L4 stage (fourth instar, hyperphagic tissue-feeding phase) - were collected from chemically protected white horse chestnut trees 1 year after imidacloprid treatment, and compared with caterpillars collected from non-treated trees in a previous study. Enzymes activity of α-amylase, disaccharidases, glycosidases and proteases was assayed. The presence of pesticide in ingested food changed the digestive enzymes profile of caterpillars. The analysis of correlations between different digestive enzymes showed many significant correlations (P<0.05) among glycolytic activities like ß-glucosidase and α-galactosidase activities. Statistically significant correlations for proteolytic activity were found between trypsin and chymotrypsin activity and aminopeptidase activity that occurred only in the 1st generation. PCA distinguished five primary components with eigenvalues higher than 1, from which the first two explain almost 59% of analyzed results. Surprisingly, in the pesticide treated groups significantly higher activities of sucrase and lactase in relation to control were found. In general, glycosidase (α-glucosidase, ß-glucosidase and ß-galactosidase) activities showed a similar pattern of activity in different generations. These results contrast with those obtained with control larvae, where significant differences in activities of α-glucosidase, ß-glucosidase and ß-galactosidase may result from the different quantity and quality food intake by subsequent generations of larvae. No inter-generation differences in total proteolytic activity were observed in treated larvae. The absolute value of total proteolytic activity was higher than that in the control group. The pesticide present in the vascular system of the horse chestnut tree significantly affected some of the digestive enzymes activities and - in consequence - also interrelationships between enzymes, what may affect the food digestion.

8-hydroxy-2'-deoxyguanosine in colorectal adenocarcinoma--is it a result of oxidative stress?

BACKGROUND: 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidatively modified lesions in DNA and is a marker of the oxidative stress. 8-OHdG is a mutagenic lesion and it can mispair with adenine, causing G:C→T: A transversion. Our task was to determine the 8-OHdG level in patients with colorectal adenocarcinoma directly in tumor tissues and corresponding normal mucosa. MATERIAL/METHODS: Samples of tumor tissues and corresponding normal mucosa of 47 patients undergoing surgery for colorectal cancer were analyzed. DNA was isolated from both tumor and normal tissues. Then, DNA was hydrolyzed to nucleotides using nuclease P1 and alkaline phosphatase. The 8-OHdG and 2'-dG (2'-deoxyguanosine) were determined in hydrolysates by high-performance liquid chromatography (HPLC) with electrochemical (EC) and UV detector. RESULTS: The levels of 8-OHdG in colorectal adenocarcinoma tissues were higher than in corresponding normal mucosa. No significant differences were shown in 8-OHdG levels in the cancerous and cancer-free tissues between age and sex and stages A/B and C/D of Duke's classification. CONCLUSIONS: 8-OHdG reflects the local oxidative stress in colon adenocarcinoma tissue together with ageing processes, but not the intensity of tumorigenesis itself. Because of many factors that could influence the oxidative modification of DNA bases, its role as a diagnostic and/or prognostic factor in colon adenocarcinoma seems to be limited.

Mobile sailing robot for automatic estimation of fish density and monitoring water quality.

INTRODUCTION: The paper presents the methodology and the algorithm developed to analyze sonar images focused on fish detection in small water bodies and measurement of their parameters: volume, depth and the GPS location. The final results are stored in a table and can be exported to any numerical environment for further analysis. MATERIAL AND METHOD: The measurement method for estimating the number of fish using the automatic robot is based on a sequential calculation of the number of occurrences of fish on the set trajectory. The data analysis from the sonar concerned automatic recognition of fish using the methods of image analysis and processing. RESULTS: Image analysis algorithm, a mobile robot together with its control in the 2.4 GHz band and full cryptographic communication with the data archiving station was developed as part of this study. For the three model fish ponds where verification of fish catches was carried out (548, 171 and 226 individuals), the measurement error for the described method was not exceeded 8%. SUMMARY: Created robot together with the developed software has features for remote work also in the variety of harsh weather and environmental conditions, is fully automated and can be remotely controlled using Internet. Designed system enables fish spatial location (GPS coordinates and the depth). The purpose of the robot is a non-invasive measurement of the number of fish in water reservoirs and a measurement of the quality of drinking water consumed by humans, especially in situations where local sources of pollution could have a significant impact on the quality of water collected for water treatment for people and when getting to these places is difficult. The systematically used robot equipped with the appropriate sensors, can be part of early warning system against the pollution of water used by humans (drinking water, natural swimming pools) which can be dangerous for their health.

Within and between seasonal changes of detoxifying capabilities of Cameraria ohridella (Lepidoptera : Gracillariidae) larvae.

Cameraria ohridella is an invasive leaf miner, a severe pest of horse chestnut trees. Chemical control needs recognition of intrinsic metabolic capability to cope with external stressing factors. Our tasks were to check annual effects of generation, and host tree age on detoxifying abilities of the last larval stage. Activities of CAT, SOD, GSTPX, GST CarE and AChE were assayed in the midgut of larvae from two localities during 3 years and three generations, annually. Activities of GSTPX and GST were high, but CAT and SOD were low in relation to other lepidopteran larvae. In general, the second generation larvae had the highest activity, indicating an effective defence against allelochemicals produced by the host. Effects of host tree age were significant for SOD, GSTPX and CarE activities. Significant annual differences on enzyme profiles of CAT, SOD, CarE and AChE reflected differences of temperature and precipitation between consecutive years.

The reproductive potential of the spiders Agelena labyrinthica and Xerolycosa nemoralis from areas contaminated with metals.

Spiders successfully colonize industrially contaminated environments and maintain relatively stable populations. The aim of this study was to explain the reproductive strategies of two spider species, Xerolycosa nemoralis (an actively hunting, sit-and-pursue predator) and Agelena labyrinthica (a web-building, sit-and-wait predator), between contaminated and uncontaminated sites. Spiders were collected from a reference site (Pilica) and two contaminated sites (Olkusz and Welnowiec). The amount of energy allocated to the eggs and the number of eggs and hatchlings as well as the hatching success were compared. Wolf spiders from the contaminated sites produced fewer but relatively energy-rich eggs, whereas web-building spiders invested their energy in the production of a higher number of less energy-rich eggs. The comparisons of the hatching percentages suggested that in the contaminated habitats, X. nemoralis achieve a hatching success similar to or higher than that of the reference population at Pilica. A. labyrinthica in the contaminated sites invested a larger amount of energy in eggs than at the reference site, but the hatching success found for this species in the contaminated areas was lower than that found at the reference site.

Differentiation of regenerative cells in the midgut epithelium of Epilachna cf nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae).

Differentiation of regenerative cells in the midgut epithelium of Epilachna cf nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae), a consumer of the Ni-hyperaccumulator Berkheya coddii (Asteracae) from South Africa, has been monitored and described. Adult specimens in various developmental phases were studied with the use of light microscopy and transmission electron microscopy. All degenerated epithelial cells are replaced by newly differentiated cells. They originate from regenerative cells which act as stem cells in the midgut epithelium. Just after pupal-adult transformation, the midgut epithelium of E. nylanderi is composed of columnar epithelial cells and isolated regenerative cells distributed among them. The regenerative cells proliferate intensively and form regenerative cell groups. In each regenerative cell group the majority of cells differentiate into new epithelial cells, while some of them still act as stem cells and persist as a reservoir of cells capable for proliferation and differentiation. Because this species is an obligate monophage of plants which accumulate nickel, proliferation and differentiation of midgut stem cells follow degeneration intensively and in a typical manner.

Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

Cellular stress reactions assessed by gender and species in spiders from areas variously polluted with heavy metals.

In the funnel web spider Agelena labyrinthica (Agelenidae; A. l.), sheet web spider Linyphia triangularis (Linyphiidae; L. t.) and wolf spider Xerolycosa nemoralis (Lycosidae; X. n.) from two differently polluted meadow sites in southern Poland, we studied the relations between antioxidant parameters (glutathione, GSH; glutathione peroxidases, GPOX, GSTPx; catalase, CAT; stress proteins-Hsp70, metallothioneins Mts), the intensity of apoptosis and necrosis, and heavy metal burdens of the midgut gland. Cellular reactions against stress caused by pollutants seemed to be sex-dependent. The concentrations of Zn and Cu in the midgut glands of male A. l. and X. n. were more than double that of the females, from both study sites. In male spiders from the heavily polluted site, both negative correlations (activity of caspase-3-like proteins vs Cu, Zn concentration; number of depolarized mitochondria vs Cu concentration) and positive correlations (number of necrotic cells vs Cu concentrations; activity of CAT vs Zn ) were noted. The defense of males against high metal content and its prooxidative effects is based mainly on GSH and CAT. In females the antioxidative reactions are species-specific and depend mainly on high peroxidase activity and on stress protein level. The increase in the number of apoptotic cells in the midgut gland of female spiders from the heavily polluted site suggests the defensive role of this process in maintaining the proper functioning of this organ.

Zinc-induced DNA damage and the distribution of metals in the brain of grasshoppers by the comet assay and micro-PIXE.

The distribution and concentration of selected elements by PIXE method and DNA damage using comet assay in brains of 1st instars of grasshoppers Chorthippus brunneus from unpolluted (Pilica) and polluted (Olkusz) site, additionally exposed to various doses of zinc during diapause or after hatching, were measured. We tried to assess the degree of possible pre-adaptation of the insects to heavy metals and evaluate the utility of these parameters in estimation of insect exposure to industrial pollutants. Additionally, the mechanism of zinc toxicity for grasshopper brains was discussed. We observed the correlation between experimental zinc dose, zinc contents in the brain and DNA damage in neuroblasts, but only in groups exposed to lower zinc concentration. For higher zinc concentration the amount of the metal in brain and DNA damage remained at the control level. Some site-related differences in DNA damage between grasshoppers from Pilica and Olkusz were observed during short-term exposure (after hatching). Significant increase in the calcium contents in the brain, proportional to zinc concentration in sand, was also observed, especially in the offsprings from Olkusz. The results may be the basis for further searching for molecular mechanisms of defense against heavy metals in insects living in polluted habitats.

Effects of dimethoate on spiders from metal pollution gradient.

In this study, an attempt has been made to assess whether a chronic exposure to metals in habitats under a strong industrial pressure might have equipped spiders with biochemical defensive mechanisms enabling them to survive an additional chemical stress. To check this, non-web-building wolf spiders Pardosa lugubris (Lycosidae) and funnel web Agelena labyrinthica (Agelenidae) were collected at five variously polluted meadows and, under laboratory conditions, intoxicated with either single or multiple dose of dimethoate (OP pesticide). Then the activities of detoxifying (carboxylesterase: CarE, glutathione S-transferase: GST), antioxidative (selene-dependent and selene-independent glutathione peroxidases: GPOX and GSTPx) enzymes as well as acetylcholinesterase as a biomarker of exposure to OP pesticides were measured. In web-building A. labyrinthica, even a single application of the pesticide caused the inhibition of CarE, GSTPx and GPOX in individuals from less polluted sites and AChE and GST in specimens pre-exposed to high metal concentrations. Multiple intoxication, irrespectively of the site, caused significant, in comparison to controls, decrease in CarE, AChE and GSTPx activities. Actively hunting P. lugubris seem more resistant to acute pesticide intoxication, since the spiders from each site had a constant level of GST and AChE. In individuals of this species from heavily polluted sites, the inhibition caused by multiple intoxication with dimethoate was stated only for glutathione peroxidases.

Joint effects of dimethoate and heavy metals on metabolic responses in a grasshopper (Chorthippus brunneus) from a heavy metals pollution gradient.

We studied how an exposure to an additional stressing factor-dimethoate, might affect detoxifying ability of grasshoppers collected at 5 meadow sites located along a heavy metal pollution gradient. Activities of esterases and enzymes linked with glutathione (GSH) metabolism were assayed 24 h after topical treatment with 0.32 microg dimethoate per insect. Inhibition of acetylcholinesterase (AChE) reaches nearly 50% of the value stated in untreated insects, without significant site-dependent differences. The pesticide also caused a significant decrease in activities of glutathione peroxidase (GPx) followed by a decrease in GSH levels in grasshoppers from all assayed groups, demonstrating high sensitivity of glutathione-dependent metabolism to the additional stressing factor. In the case of glutathione reductase (GR) and carboxylesterases (CarE) the fall of activity was shown especially in insects from less polluted meadows and the reference site. Glutathione reductase (GR) activity in individuals treated with dimethoate did not decrease only in insects from the most contaminated site I. This might suggest the trade-off mechanisms adapting grasshoppers to life in seriously polluted environments.

Relations between metals (Zn, Pb, Cd and Cu) and glutathione-dependent detoxifying enzymes in spiders from a heavy metal pollution gradient.

We studied the relations between glutathione-dependent detoxifying enzymes and heavy metal burdens in the web-building spider Agelena labyrinthica (Agelenidae) and the wolf spider Pardosa lugubris (Lycosidae) from five meadow sites along a heavy metal pollution gradient. We assayed the activity of glutathione-S-transferase (GST) and glutathione peroxidases (GPOX, GSTPx), and glutathione (GSH) levels in both sexes. Except for GSH vs Pb content, we found significant correlations between GPOX and GSTPx activity and metal concentrations in females of A. labyrinthica. The highest activity of these enzymes measured in the web-building spiders was found in the individuals from the most polluted sites. In P. lugubris males significant correlations were found between GST and Pb and Zn concentrations, and between GPOX and GSTPx and the concentration of Cu. GST activity was higher in males collected from less polluted areas. Thus, detoxifying strategies against pollutants seemed to be sex-dependent. Actively hunting spiders had higher metal concentrations, maintaining lower activity of detoxifying enzymes and a lower glutathione level.

Profiles of enzymatic activity in earthworms from zinc, lead and cadmium polluted areas near Olkusz (Poland).

The aim of the study was to determine whether there are signs of adaptation of soil fauna to a gradient of heavy metal contamination. Earthworms Aporrectodea caliginosa, Lumbricus terrestris and Eisenia fetida were collected during the spring and summer of 2000 and 2001 from meadow sites situated between 2 and 32 km from the Bukowno-Olkusz complex of zinc-lead ore mines and smelters. The heavy metal content in the soil near smelters reaches 10,500 mg/kg (d.w.) for Zn, 2600 mg/kg for Pb and 81.9 mg/kg for Cd. The sites differ with respect to species composition of earthworm community, with A. caliginosa being dominant. Complete data was obtained only for A. caliginosa, since other species were not abundant at all investigated sites during the whole period of investigation. The body burdens of Zn, Pb, Cd and Cu in A. caliginosa reached 1500, 100, 220 and 10 microg/g, respectively, in the vicinity of the smelter (2-4 km), and decreased to 400, 2, 36 and 6 microg/g at the most distant site (32 km). Cadmium and lead content was significantly elevated in the whole body of L. terrestris collected at the site 2.5 km distant from the smelters when compared to more distant sites, while in E. fetida only the body burden of cadmium was elevated at the nearest site compared to the next site of transect. Activities of glutathione peroxidase (GPX; EC 1.11.1.9) against hydrogen peroxide (H2O2) or cumene hydroperoxide (cumOOH), glutathione reductase (GR; EC 1.6.4.2), glutathione S-transferase (GST; EC 2.5.1.18) and catalase (CAT; EC 1.11.1.6) were assayed in postmitochondrial supernatant obtained from whole body homogenates. Seasonal and annual variations of enzyme activity were reflected by higher GPX activity in the late summer of 2001 in comparison with the spring and summer of 2000. This may reflect severe drought in the spring and summer of 2000. The activity of both GPX isozymes, GR and GST in A. caliginosa and L. terrestris increased with increasing distance from the smelters and reached maximum at sites III and IV (4 and 8 km from the smelters, respectively) and then it decreased in the animals from site V (32 km). These may be the effects of antagonism between the enzyme inducing and enzyme inhibiting action of smelter emissions, a phenomenon known as a hormetic effect. It is postulated here that this effect is of diagnostic value for metal pollution biomonitoring.