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The novel program for graphical display and editing of molecular systems, luscus, is described. The program allows fast and easy building and/or editing different molecular structures, up to several thousands of atoms large. Luscus is able to visualise dipole moments, normal modes, molecular orbitals, electron densities and electrostatic potentials. In addition, simple geometrical objects can be rendered in order to reveal a geometrical feature or a physical quantity. The program is developed as a graphical interface for the MOLCAS program package, however its adaptive nature makes possible to use luscus with other computational program packages and chemical formats. All data files are opened via simple plug-ins which makes easy to implement a new file format in luscus. The easiness of editing molecular geometries makes luscus suitable for teaching students chemical concepts and molecular modelling.

High density brines are concentrated salt solutions which are regularly used to control the hydrostatic pressure in oil wells during special operations in exploration and production of natural gas and crude oil [1]. Calcium, sodium, potassium, magnesium and zinc chloride and bromide are most usually used as workover high density fluids, in quantity of 5 – 20 m3 per well [2]. Various concentrations of high density brines are left in the mud pits nearby the drilling site as waste and may pollute fresh and ground waters by spilling over and drain off through the ground. Investigating the effects of that kind of pollution was necessary, so a more extensive laboratory research of the toxic effects of high density brines on several test organisms (Lemna minor L., Daphnia magna L. and Allium test) was carried out before. The research results of the effect of sublethal concentrations of calcium chloride and calcium bromide and their mixtures 1:1 on the survival, growth and fecundity of the freshwater snail Planorbarius corneus L. showed a considerable dose-response decline of the survival, growth and fecundity [3]. In this study the effect of lethal concentrations of calcium chloride and calcium bromide (5985 ppm CaCl2 and 7939 ppm CaBr2) on the tissues of Planorbarius corneus L. was investigated. Histological samples were prepared by fixing the tissues with Bouin's solution (saturated picric acid, glacial ethanoic acid and formalin), imbedding in paraffin and sectioning between 6-8 μm. Samples were then stained using hemalaun-eosin [4]. The prepared samples showed no obvious histopathological changes except widening of hemolymphatic area in some organs (excretory organ, mantle cavity, osphradium, salivary gland) in comparison to the control (Figures 1 and 2). It was concluded that hyperosmotic medium caused a higher hemolymph viscosity and slower flow thus accumulating hemolymph in the hemolymphatic area and causing observed expansions.

Silicon is one of the most frequent elements in the Earth's crust. It is an essential element in lower life forms. The effect of silicon on aluminium intake in the body was investigated more recently. Presence of silicon in plant cell culture reduces the death rate compared to cells treated only with aluminium. Small amounts of silicon have a positive effect on the proliferation and differentiation of human osteoblasts while higher concetrations stimulate osteoblasts to cell death. Some authors reported that higher concentrations of silicon damage DNA, and is therefore used in vitro as an inducer of cancer. We analyzed the effect of sublethal concentrations of silicon (0.17 g/dm3, 0.20 g/dm3, 0.23 g/dm3, 0.25 g/dm3, 0.27 g/dm3, 0.30 g/dm3) on Polycelis felina (Daly.) in laboratory conditions. Animals were left in testsolutions for 24 hours at 4 degree. After single exposure animals were transferred into clean aquarium water for recovery and were observed following 15 days. It has been established that sodium metasilicate, depending of dosage of applied concentration, causes mortality, decephalization, depigmentation morphological and cyto - histological changes. Mortality rate and the most intensive morphological and cyto - histological damages were established on the second and third day after the treatment. On histological slides of treated planarians were visible damages of basal membrane and parenchymal cells. Using HPLC a great amount of silicone was found in the body tissue so we concluded that silicon in increased doses had a damaging effect on Polycelis felina (Daly.).

Green hydra (Hydra viridissima Pallas, 1766) is a fine classical example of endosymbiotic organism. In its gastrodermal myoepithelial cells it contains unicellular photoautotrophic algae. Microalgae have a significant role as primary producers in aquatic ecosystems and are the basis of many food chains. For the first time performed was test of comparative toxicity of aluminium and norflurazon on isolated algae from green hydra, Desmodesmus subspicatus (Chodat) Hegewald et Schmidt and Mychonastes homosphaera (Skuja) Kalina et Puncochárová, which belong to the group Chlorella zagrebiensis Kovac. et Jelen. and on related free-living species Chlorella kessleri Fott et Novak. [K&H, 1992] strain LARG/1 and Chlorella vulgaris Beij. [K&H, 1992] strain SAG 211 - 11b. From evolutionary and ecotoxicological point of view, researched were cell viability, division, shape and size, level of damage, chloroplast fragmentation, intensity of green color, in relation to the concentrations of toxicants used in the experiment, by use of Chlorella test, light microscopy and morphometry. Today it is known that algal endosymbiotic partner from green hydra can be maintained in a permanent stable lab culture, so the special attention was pointed towards researching the differences between viability of isolated endosymbionts and their free-living relatives. The algae isolated from green hydra survived to the less extent the unfavorable conditions of the microenvironment than their free-living relatives, which showed greater viability and lower level of damages. It seems that separation of endosymbiotic partners left trace on the viability of isolated species. Once isolated, former endosymbiotic algal partner is less adapted to the new microenvironment than its free-living relatives.

Iron is among the mo t common metals in Earth's crust. Hydra is a freshwater animal that belongs to the phylum Cnidaria. Toxic effect of iron on various organisms has been researched before but this is the first documented research on its effect on hydras. Green hydra contains symbiotic algae in its gastrodermal myoepithelial cells. Individuals of symbiotic green hydra were treated with six concentrations of aqueous solution of iron dextran, while aposymbiotic brown hydras were treated with 14 concentrations of aqueous solution of iron dextran ([C6Hlo03ln[Fe(OH)3]m) for three days. The following morphological changes were monitored by stereolupe: migration of individuals in experimental dish, changes of body shape, reaction to mechanical stimuli, tentacle reduction, mucous secretion and asexual reproduction. Migration of hydras was the first step in evading deleterious microenvironmental conditions, followed by changing its body shape. AI lower concentrations of iron some green hydra individuals budded, expressing the horme tic effect. The secretion of mucous, a mechanism for removal of toxins, was observed. High concentrations of iron caused morphological and histological changes and mortality. Histological changes were observed by light microscope Nikon Eclipse E600 and software for morphometric analysis of histological images LUCIA G 4.80 Morphometric analysis included measuring of endoderm and ga troderm segments area and mesoglea thickness. For histochemical detection of iron depo itions in hydra tissue, we used Pearls' Prussian Blue method. Iron depositions in hydra tissue were observed as blue staining within cells (Figure 1). Thickness of mesoglea (jlm) was determined morphometrically by randomly choosing 150 different spots of variou thicknesses at iron concentrations of I mg/L, 400 mg/L, 1400 mg/L and 1500 mg/L, and in control group. Surface of 15 ectoderm and gastroderm segments (J.lm2) measuring 100 jlm in length, at same concentrations of iron was also measured. Iron was deposited in ectoderm and gastroderm of hydras. Damage to the cell layers increased with increasing concentrations of iron. There were no iron depositions in mesoglea, but its thickness increased during the treatment. Green hydra showed a higher level of resistance in surviving the unfavorable conditions of microenvironment than brown hydra. Symbiosis was pointed out as an evolutionary advantage.

U radu su opisani postojeći alati za generiranje sintetičkog mrežnog prometa te za ponavljanje snimljenog mrežnog prometa. Predloženi su načini kako bi se izlaz tih alata mogao direktno ubacivati u različite dijelove jezgre operacijskog sustava Linux za potrebe ispitivanja IPTables pravila.

In this research we studied how different growth media for hydras affect endosimbiotic algae present in green hydra (Hydra viridissima Pallas, 1766) (Figure 1). We used three standard solutions for hydra growth prepared in aerated water: CN (NaHCO3, CaCl2), KNC (NaHCO3, KCl, CaCl2) and M (tris-HCl, NaHCO3, KCl, MgCl2, CaCl2) [1]. Dilution of all the solutions was 1/40. We placed 10 hydras in each of the solutions, one group in the glass-window refrigerator and the other at the room temperature. The experiment lasted for five days after which period algae were extracted by maceration (Figure 2). By light microscope Nikon Eclipse E600 we performed morphological observations: division, shape, color and cloroplasts of the algal cells. Micrographs were made by digital camera Nikon DXM1200. In CN and KNC solutions cell divisons to up to three daughter cells were observed and also a wide range of cell sizes. Chlorplasts were of regular, cup-shaped form. Algae were regularly round-shaped and green. In M solution the most successful result in terms of cell division where cells divided to up to four daughter cells was noticed. Chloroplasts seemed to have some irregularities in the apperance (dot-like inclusions). Cells seem to appear green-yellow and were regular in shape (Figures 3 and 4). It seems that the room teperature in general showed better conditions for endosymbiotic agal growth. Based on the results obtained, we concluded that endosymbiotic algae managed to grow best in the M solution, although cells seemed to change the color. In the end all the three culture solutions showed benefit for algal growth and maintenance of hydra. For comparison, the best solution for growing green hydra was KNC solution (1:20) where budding was recorded. After these preliminary obeservations more detailed light microscopy including morphometry as well as electron microscopy analysis should be performed to reveal more details.

The sequential product ion (MSn) fragmentation of four symmetric diamide derivatives of 3, 4-ethylenedioxythiophene were characterized using ion trap mass spectrometry with electrospray ionization and their fragmentation patterns were studied. The experimental data consists of mass spectra obtained by tandem mass spectrometry, and calculations were obtained by M06-2X/6-31G (d, p) method. Investigated compounds represent building blocks in synthesis of compounds used in different area of chemistry and industry such as in medicinal chemistry, as potential anticancer and anticonvulsant agents, in organic chemistry as linkers for solid-phase synthesis, in synthesis of variety of materials in polymer chemistry. We present herein the investigation of fragmentation pathway of protonated diamide derivatives of 3, 4-ethylenedioxythiophene that involves the identification of fragments, influence of proton transfer on direction of fragmentation and mechanisms of reactions by which fragmentation process occurs. Data obtained from product ion spectra of these protonated compounds and DFT calculations indicate that the fragmentation process takes place via the four main reactions: amido-iminol proton transfer, reverse cycloaddition, cleavage of amide bond, and isocyanic acid elimination. The 3, 4- ethylenedioxythiophene-2, 5-dicarboxamide was observed as intermediate in the fragmentation of its alkyl derivatives. To our knowledge, this work brings the first correct description of the mechanism of elimination of isocyanic acid.

Flavonoids are phenolic compounds widely present in plants as secondary metabolites. They exhibit a wide range of biological effects arising mainly from their ability to modulate several enzymes or nuclear receptors, alter gene expression, and chelate bivalent metals. Quercetin is the most abundant of the flavonoid molecules in plants. We studied the effect of quercetin on the multi-eyed flatworm Polycelis felina (Daly.) in laboratory conditions. This is a very suitable test-organism availabe year-round in nature, easily maintained in the laboratory. The results showed that depending on the dose and treatment period quercetin caused locomotory (behavioral) changes of treated animals such as aggregating into groups, resting and unsynchronized movements and twisting of particular body parts. Morphological changes in the form of depigmentation of certain body parts were the result of damage or deterioration of reticular and parenchymal cells. On histological preparations in the period between the third and seventh day after the treatment in gastroderm were determined numerous basophilic bodies that represented cell parts of damaged and decomposed cells. All histological preparations revealed a larger decline of cellular material, therefore a damaging effect of quercetin on the multi-eyed flatworm Polycelis felina (Daly.). The most exceptional changes are noticed on totipotent cells of neoblasts and reticular cells. Determined changes were most intensive on the third day after the treatment, when animals were in the process of recovery and above mentioned cells were represented in higher number compared to control group of animals.

One of the most frequent metalloid and metallic elements in the Earth's crust are silicon and aluminium. Silicon is an essential element in lower life forms and its biological function in higher forms of life is not known. Acidification of water ecosystems and lowering the pH value increases solubility of metal ions e. g. aluminium, which at higher concetrations becomes toxic. Aluminium is a known neurotoxin, which interferes with the synthesis of neurotransmitters in the central nervous system of mammals. We analyzed the effect of aluminium (80 mg/dm3, 100 mg/dm3) in neutral and acidic media, pH 7.1 and 5.2 and sublethal doses of silicon (0.17 g/dm3, 0.20 g/dm3, 0.23 g/dm3, 0.25 g/dm3, 0.27 g/dm3, 0.30 g/dm3) on Polycelis felina (Daly.) respectively. It has been established the toxic effect of aluminium in applied concentrations upon planarian species. Aluminium in acid media induced higher mortality, stronger morphological and behavioural changes and more extensive tissue damage. Also silicon, depending of dosage of applied concentration, causes mortality, decephalization, depigmentation, morphological and cyto - histological changes. Using HPLC a great amount of silicon was found in the body tissue. Most intensive damages were established on the first and second day after the treatment in the experiment with aluminium in acidic media and on the second and third day after the treatment in the experiment with silicon. On histological slides of treated planarians with aluminum and silicon were visible same changes: damages to the epidermis, degradation of tissue, decomposition of mucous layer and an increased number of rabdites and neoblasts.

U navigaciji sve je veća potražnja za točnim, cijenom prihvatljivim ali pouzdanim sustavima. Jedan od takvih sustava je žiroskop, čiju primjenu na brodu nalazimo u obliku žiro kompasa. Mehanički žiro kompas kao takav, ima svoje greške (otklon glavne osi rotacije od pravog meridijana) koje ovise o kursu broda i brzini njegove promjene, brzini broda, zemljopisnoj širini, itd. S razvojem tehnologije i otkrićem lasera, a u cilju stvaranja točnijih i pouzdanijih kompasa, nastala je nova generacija žiro kompasa temeljena na Sagnacovom efektu, tj. laserski žiro kompasi. Također su kasnije otkrićem optičkih vlakana nastali žiro kompasi koji kao vodič laserske zrake koriste optička vlakna.

Hydras are simple freshwater invertebrates, members of the phylum Cnidaria, class Hydrozoa, order Hydroida, family Hydridae. Green hydra (Hydra viridissima Pallas, 1766) is a classical example of symbiosis. It forms an endosymbiotic relationship with individuals of unicellular green algae that reside in the gastrodermal myoepithelial cells of hydra. Each alga is surrounded by perialgal space and placed in a separate vacuole symbiosome. Ultrastructural modifications of green hydra symbiosis in an unfavorable environment perform as protective mechanisms in green hydra symbiosis and point towards a certain degree of endosymbiont independency (Figure 1). In this work quantitative characterization was performed (Figure 2). Individuals of green hydra (strain S1J-J1) were used for the experiment in the laboratory conditions. Ultrastructural changes were observed by means of TEM. For TEM standard preparation methods were used. Hydras were fixed in 2 % glutaraldehyde. After post-fixation the material was dehydrated through an ethanol series, embedded into resin and contrasted with uranyl-acetate and Pb-citrate. Micrographs were obtained using the Zeiss EM10A and FEI Morgagni 269D electron microscopes. After the treatment with norflurazon and cinoxacin and period of recovery after the treatment with norflurazon, morphometrically were worked out the following parameters: width of perialgal spaces, area and perimeter of symbiosomes. In all the parameters numerical values were changed according to the control. The numerical values of the three measured parameters showed oscillations in the size of perialgal spaces and symbiosomes (Figure 2), as the result of disturbance of symbiosis. Damages were often so huge that the measurements could not be performed, because the values would be endlessly high. It could be supposed that in the longer period of recovery the result would be closer to the control values. After the recovery period a shift towards the measured values was traced, in relation to the impossibility of measurement of endless values in the treatments themselves. A little is known about the molecular basis of survival of endosymbiotic algae inside the vacuoles and the control of interactions of both partners. This opens possibilities for further investigations on mechanisms of green hydra symbiosis.

Istraživanje metabolizma potencijalnih lijekova važno je zbog određivanja brzine, i mehanizma biorazgradnje, te kemijske strukture nastalih produkata. Od iznimne je važnosti odrediti aktivne metabolite osobito u svrhu priprave prolijekova, kao i odrediti potencijalno toksične spojeve koji izazivaju neželjene sporedne reakcije u organizmu. Pojedini strukturni dijelovi (potencijalnih) lijekova direktno utječu na brzinu metabolizacije i fizikalno-kemijska svojstva dobivenih metabolita, a time i na biološku aktivnost. U ovom je radu prikazana korelacija eksperimentalnih podataka i teorijskih računa energija i duljina veza kod serije amidnih derivata 3, 4-etilendioksitiofena, te je proučavan utjecaj supstituenta na fragmentaciju amidne skupine. Eksperimentalni podaci sastoje se od spektara masa dobivenih tandemskom spektrometrijom mase pri različitim kolizijskim energijama, a računi su dobiveni M06-2X/6-31G(d, p) metodom. Računskim metodama određen je fragmentacijski mehanizam ispitivanih spojeva i nađeno je da je u skladu s eksperimentalnim rezultatima. Zaključeno je da je iz eksperimentalnih podataka moguće povući zaključke o relativnim energijama veza.

Za određivanje udaljenosti u pomorskoj navigaciji danas se najčešće koriste radari ili laserski daljinomjeri. Međutim, osim elektroničkih pomagala do udaljenosti se može doći korištenjem klasičnih navigacijskih pomagala, npr. preko horizontalnih kutova određenih sekstantom ili smjernom pločom, pojavom/iščeznućem objekta poznate visine na horizontu, mjerenjem vertikalnih kutova uz pomoć sekstanta, itd. Određivanje udaljenosti uz pomoć vertikalnih kutova objekata čija je visina poznata u osnovi je jednostavna metoda, međutim zbog niza mogućih grešaka i nepouzdane stajnice u praksi se rijetko koristi.

Matematički modeli širenja i suzbijanja infekcije pokazuju da bi najučinkovitija mjera bila liječenje svih AIDS/HIV inficiranih, no ta je mogućnost s obzirom na sve okolnosti u svijetu, malo vjerojatna.

In metal oxide semiconductor capacitors subjected to electrical stress the dielectric breakdown is due to the creation of new electronic traps, forming a conductive path between the gate electrode and substrate. The breakdown phenomenon depends on the dielectric thickness and the number and the nature of traps needed to form a perocative path across the dielectric. Here we tried an approach to modeling the dielectric degradation based on fractal distribution of traps. The fractal pattern of cluster of traps formation is produced by algorithm based on difussion limited aggregation (DLA) model, used in the literature to model irreversible colloidal aggregation. In our approach trap generation and migration is not random and we introduced several parameters that simulates the realistic situation. The results obtained by this model are compared with the Weibull statistics used to desccribe the dielectric film failure. Finally the results of the modelling are compared with the experimental data obtained on thin dielectrc film breakdown.

Green hydra (Hydra viridissima Pallas, 1766) is a classical example of endosymbiosis. Described ultrastructural features of green hydra symbiosis are extensive widening of perialgal spaces, degradation, loss and fusion of symbiosomes. These changes are considered as defensive and protective mechanisms and the endosymbionts show greater viability than their host. Also, a certain degree of independency was noticed and for the first time endosymbiotic algae from green hydra have been successfully isolated and permanently maintained in pure lab culture. Microscopical and molecular analyses of the isolated endosymbionts resulted in the discovery of two hitherto undescribed endosymbiotic species in green hydra. In final conclusion, endosymbiotic algae from green hydra perform as the “stronger” symbiotic partner in green hydra symbiosis. We keep on searching for more evidence and results.

Geometrije predreakcijskih kompleksa, prijelaznih stanja i produkata za reakciju adicije OH radikala na fluorbenzen i klorbenzen su optimirane MP2/6- 31+G(d, p) računom. Geometrijski parametri predreakcijskih kompleksa, prijelaznih stanja i produkata za reakciju OH radikala na heksafluorbenzen i heksaklorbenzen su optimirani MP2/6-311G(d, p) računom. Anharmoničke vibracije heksafluorbenzena i heksaklorbenzena su dobivene rješavanjem Schrödingerove jednadžbe na potencijalu koji je izračunat za te vibracije. Rotacijske energije OH radikala u kompleksu s heksafluorbenzenom i heksaklorbenzenom su također određene rješavanjem Schrödingerove jednadžbe na potencijalu izračunatom MP2/6-311G(d, p) računom. Energijski parametri reakcija su određeni G3 računom. Ukupne konstante brzine reakcija su određene računanjem konstante brzine disocijacije predreakcijskog kompleksa RRKM metodom, konstante ravnoteže za stvaranje predreakcijskog kompleksa statističkom termodinamikom i udjela reakcijskih produkata u reakcijskoj smjesi Gillespie-vom stohastičkom metodom. Pogreška, nastala uslijed opisivanja relativne translacije OH radikala u predreakcijskim kompleksima harmoničkim oscilatorom je provjerena opisivanjem tog gibanja modelom čestice u kutiji. Pogreška u izračunatoj energiji je provjerena modificiranjem energijskih parametara za prihvaćenu srednju apsolutnu pogrešku G3 računa.

For the first time effects of norflurazon on green (Hydra viridissima Pallas, 1766) and brown hydra (Hydra oligactis Pallas, 1766) in high- and low-light conditions were investigated in order to establish the extent of damage that this substance inflicts, with special emphasis on the "bleaching effect" and the effect on hydra-algae symbiosis. Green hydra is a typical example of an endosymbiotic organism. The gastrodermal myoepithelial cells of green hydra contain endosymbiotic algae. Norflurazon is a selective translocational herbicide that induces a "bleaching effect" on newly developed chloroplasts, resulting in a decrease of photosynthetic activity and viability of the organism. In the experiment, morphological (binocular), cytological and histological (Bouin fixative, dehydration, paraplast embedding, Hämalaun-eosine staining, light microscope) and conventional transmission electron microscopy (cTEM) (glutaraldehyde, dehydration, raisin, uranyl-acetate, Pb-citrate) were used. Depending on the concentration and light conditions, norflurazon caused mortality, deformations, changes in behavior, locomotion and asexual reproduction, changes in the structure of all 3 layers in hydras, changes in the position and shape of endosymbiotic algae inside the hydra body as well as ultrastructural changes of treated hydras and endosymbiotic algae. Under low concentrations of norflurazon the effects on hydra were similar to the controls, while in the highest concentrations especially manifested were antichloroplastal and antimitochondrial effects. Norflurazon caused a great extent of damage and induced deleterious effects also upon other cellular components such as cellular membranes, ER, Golgi apparatus, ribosomes. Newly developed buds in symbiotic green hydras were not bleached. After a recovery period, the green hydra individuals that had survived re-established regular endosymbiosis with algae and recovered completely, whereas brown hydras recovered only partially.

Symbiotic associations are of a wide significance in evolution and biodiversity. The green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells it harbors the individuals of a unicellular green algae. Thus far it has been accepted that endosymbiotic algae from green hydra are impossible to grow in a clean lab culture. Hydras were collected on two distant localities in Zagreb, Croatia: Jarun Lake (strain S1J - J1) and the greenhouse of the Botanical gardens (strain S1B - S1) from the surface of submersed plants, and maintained in lab cultures. In this study endosymbiotic algae from green hydra have been successfully isolated and permanently maintained in a stable clean lab culture on a sterile deep stock agar in tubes by means of microbiological methods. We reconstructed the phylogeny of endosymbiotic algae using the 18S rRNA gene, which is unavoidably used in the phylogeny of symbiotic organisms as essential molecular marker which evolves slowly and is functionally preserved, to clarify its current status and to validate the traditional inclusion of these endosymbiotic algae within the Chlorella genus. Molecular analyses (Bayesian analysis, Maximum likelihood, Maximum parsimony) established that different genera and species of unicellular green algae could be present as symbionts in green hydra, depending on the natural habitat of a particular strain of green hydra. The results showed that the isolated endosymbionts belonged to the genera Desmodesmus and Mychonastes. The green hydra symbiosis revealed that hydra-alga associations’ biodiversity is much more complex than previously thought.

Hydras are simple aquatic organisms, members of the phylum Cnidaria, usually found in unpolluted freshwater. Green hydra (Hydra viridissima Pallas, 1766) is an endosymbiotic and brown hydra (Hydra oligactis Pallas, 1766) is a non-symbiotic species [1]. Aluminium is one of the most abundant elements in the Earth's crust, but its effects upon living organisms have not been much studied. The aim of this research was to explore the effects of aluminium in two closely related hydra species, and to trace the extent of damage caused. For the first time, a modified alkaline comet assay [2] was used to study primary DNA damage in green and brown hydra cells. Five animals of each species were treated under laboratory conditions (21.5°C) with aluminium-sulphate (Al2(SO4)3.18H2O ; 25, 250, 475 mg/L) in aerated aquarium water in glass dishes, 6 cm in diameter, 3.5 cm in height (photoperiod 10 hrs light/15 μ mol/m2s, 14 hrs dark) and compared with the control groups. After 72 hrs, animals from each of the concentrations of Al were transferred into a micro-test-tube containing 10 μ l of chilled distilled water. Hydras were homogenized with a 100 μ l micropipette by sucking the animals in and out several times. Fully frosted slides were coated with 1% and 0.6% normal melting point (NMP) agarose. Homogenized hydras were mixed with 0.5% low melting point (LMP) agarose and placed on the slides. The slides were immersed for 1.5 hrs in freshly prepared ice-cold detergent lysis solution pH 10. Alkaline denaturation was carried out in freshly prepared buffer pH 13.0 for 15 min and electrophoresis for another 10 min at 0.8 V/cm. Slides were neutralized with Tris-HCl buffer pH 7.5, stained with ethidium bromide and examined using epifluorescence microscope Orthoplane (Leitz Wetzlar, Germany), equipped with immersion objective 170/0.17 63/1.30, an excitation filter of 515– 560 nm and a barrier filter of 590 nm (Leitz Wetzlar, Germany). A total of 100 comets were scored per slide. Using a black and white high performance CCD camera 4900 Series (COHU), the microscope image was taken and analysed by Comet Assay II software (Perceptive Instruments Ltd.). Tail length (μ m) and tail intensity were used as a measure of DNA damage. Entire DNA damage evaluation was performed in duplicate. Means of repeated experiments were used to test the difference of the tail length and intensity values between treated and control animals applying the Student’ s t-test (Statistica 5.0, StatSoft, USA). The dependence of the comet assay endpoints on the Al concentration was evaluated by calculating the Spearman correlation coefficient. Multiple regression was performed to test the predictivity of the Al-dose-response. Significance level was set to 0.05. Aluminium toxicity triggered DNA changes. DNA tail length and intensity changes were greater in brown than in green hydras. Since the comet assay detects DNA lesions at a single cell level [3], it enables to observe intercellular differences in the genomic susceptibility. Control cells appear as compact orange spheres, whereas cells bearing the genome damage exhibit more or less dense tail of orange spots resembling the comets in the sky (Fig. 1). Brown hydra was more susceptible to aluminium than green hydra, confirming the evolutionary advantage provided by symbiosis. Due to its higher genetic susceptibility, brown hydra appears to be a better biomarker for aquatic toxicology studies compared with green hydra. Due to its higher correlation and predictivity, the tail intensity was shown to be a more relevant comet assay parameter for DNA damage than tail length. Biomonitoring protocols using hydra and the comet assay could be developed to provide a valuable and rapid method for determining the quality of freshwater. 1. Douglas, A.E., Symbiotic Interactions. Oxford University Press Inc., Oxford & New York (1994) 2. Singh, N.P. et al., Exp. Cell Res. 175 (1988) p184. 3. Collins, A.R., Mol. Biotechnol. 26 (2004) p249. 4. The presented results are a product of a scientific projects “ Molecular phylogeny, evolution and symbiosis of freshwater invertebrates” and “ Genotoxicity of chemical and physical agents of natural and anthropogenic origin” carried out with the support of the Ministry of Science, Education and Sport of the Republic of Croatia. We thank Professor D.H.S. Richardson and the two reviewers for helpful comments on this manuscript. AB Figure 1. Micrographs of green hydra cell (a) and brown hydra cell (b) with highly damaged DNA. Hydras were exposed to the 250 mg/L Al2(SO4)3.18H2O for 72 hours. Micrographs were taken using Comet Assay II software and COHU CCD camera 4900 Series. Magnification 400x.

News on green hydra endosymbiosis are described: the possibility of isolation of unicellular photoautotrophic endosymibionts, their taxonomic identity, their morphometry and relationship to free-living relatives, as well as the mechnisms of symbiosis re-assembly, after the symbiosis breakdown.

Symbiotic associations are of a wide significance in evolution and biodiversity. Symbiosis is a long-term close relationship between two or more species which belong to differently named categories. Green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells it harbors individuals of unicellular green algae. Each alga is placed inside one vacuolar membrane forming the symbiosome. Symbiogenesis denotes new form, function or metabolic capability that one symbiotic partner gained from the other. Newly described mechanisms of the hydra-alga symbiosis are widening of perialgal space, degradation and loss of symbiosome, fusion of symbiosomes and perialgal spaces, and their role in regular re-assembling of the symbiosis (1). For the first time endosymbiotic algae from green hydra have been successfully isolated and permanently maintained in a clean lab culture. By microscopical, morphometrical and molecular analyses it was established that different genera of unicellular green algae could be present as symbionts in green hydra, only one at a time. Endosymbiotic alga performed as the “ stronger” symbiotic partner in green hydra symbiosis, but once isolated, endosymbiotic alga proved to be less resistant to environmental conditions than the aposymbiotic one. The results referred to the fact that symbiogenesis in green hydra has probably not been finished yet.

Simbiotske asocijacije imaju široki značaj u evoluciji i bioraznolikosti. Simbioza je dugotrajan tijesan suživot dvaju ili više vrsta organizama. Zelena hidra je tipičan primjer endosimbioze, jer u svojim gastrodermalnim mioepitelnim stanicama sadrži jednostanične zelene alge. Novoopisani mehanizmi simbioze hidre i alge nakon djelovanja toksikanata su širenje perialgalnog prostora, degradacija i gubitak simbiosoma, spajanje simbiosoma i perialgalnih prostora, te njihova uloga u ponovnoj pravilnoj uspostavi simbioze. Po prvi puta su uspješno izolirane i trajno održavane u čistim laboratorijskim kulturama endosimbiotske alge iz zelene hidre. Mikroskopskim, morfometrijskim i molekularno biološkim metodama utvrđeno je da različite vrste i rodovi jednostaničnih zelenih algi mogu biti simbionti u zelenoj hidri, ovisno o prirodnom staništu pojedinog soja zelene hidre. Alge promjenjive morfologije pripadaju rodovima Desmodesmus i Mychonastes. Izvršeno je taksonomsko razrješavanje pitanja pripadnosti tih algi, koje je do sada bilo nerazjašnjeno i neriješeno. Za endosimbiotske zelene alge iz zelene hidre predlaže se naziv Chlorella zagrabiensis Kovac. i Jelen. (2007). S evolucijskog i ekotoksikološkog stanovišta po prvi puta je praćen i uspoređivan učinak aluminija na test organizme koji dijele složene evolucijske i simbiotske odnose: zelena (Hydra viridissima Pallas, 1766) i smeđa hidra (Hydra oligactis Pallas, 1766), te zelena alga Chlorella kessleri Fott i Novak. [K&H, 1992] i endosimbiotska alga izolirana iz zelene hidre. U tu svrhu po prvi puta su na hidrama korištene posebno prilagođene metode bojenja eriokromovim cijaninom i komet tehnike. Aluminij je uzrokovao morfološke, citološke, histološke i lokomotorne promjene te smrtnost, hormestički učinak i oštećenja DNA. Iskazani su oblik i distribucija te površina depozicija aluminija u odnosu na stanice koje ih sadrže. Pokazan je odnos oštećenja stopala hidre i vijabilnosti organizma. Utvrđeno je da aluminij ne predstavlja okolišnu opasnost za populacije hidri i algi u prirodi, ali je pokazan mogući doseg njegova djelovanja u okolišu. Kao krajnji rezultat narušene simbioze hidre i alge pokazala se ponovna uspostava endosimbioze, oporavak zelene te djelomični oporavak smeđe hidre. Zelena hidra pokazala se otpornijom od smeđe hidre, a izolirana endosimbiotska alga u odnosu na aposimbiotsku manje otpornom. Kao “ jači” simbiotski partner u zelenoj hidri utvrđena je endosimbiotska alga. Rezultati upućuju na činjenicu da simbiogeneza u zelenoj hidri vjerojatno nije u potpunosti završena.

Simbiotske asocijacije od velikog su značaja za evoluciju i biodiverzitet. Zelena hidra je klasičan primjer endosimbioze jer u svojim gastrodermalnim mioepitelnim stanicama sadrži jednostanične zelene alge. Prvi puta u sklopu ovog istraživanja uspješno je izolirana i održavana u trajnoj laboratorijskoj kulturi endosimbiontska alga iz zelene hidre na osnovi čega je rekonstruirana njena filogenija. U istraživanjima filogenije simbiontskih organizama gen za 18S rRNA se koristi neizostavno kao prikladan, visoko konzerviran molekulski marker, koji sporo evoluira i funkcionalno je očuvan. U našem istraživanju rekonstruirali smo filogeniju endosimbiontske alge iz zelene hidre upotrebom gena za 18S rRNA kako bi razjasnili taksonomski status endosimbionta i utvrdili da li se radi o algi roda Chlorella. Molekularno filogenetske analize ukupno 41 sekvence iz našeg istraživanja i GenBank baze sekvenci pokazale su da naši uzorci endosimbiontskih algi iz zelene hidre ne pripadaju rodu Chlorella, već rodu Desmodesmus, najvjerojatnije vrsti Desmodesmus subspicatus.

Background and Purpose: Chlorella test was performed for the first time. The interesting application of a newly proposed test in ecotoxicological research was explained. It was tested using the herbicide norflurazon as bleaching toxicant. The experimental object was green alga Chlorella kessleri Fott et Novak. [K&H, 1992]. Materials and Methods: Algae were treated on the surface of deep stock agar in tubes. Instead of dH2O t the equivalentmeasure of aqueous solution of toxicant was used in the medium. This is the essence of the Chlorella test method. Results: Already after a short period of two days, morphological changes were distinguished macroscopically. Bleaching of algae was present in particular tubes, as the result of the effect of the toxicant upon the algal growth and viability. Conclusions: We propose Chlorella test as a toxicity test that provides easy detection of sublethal, lethal and NOEC. The use of this quick, simple, indicative, cheap and applicative test is proposed as amethod for research on the preliminary effects of environmental pollutants upon green algae in culture.

Hydras are simple aquatic organisms, members of the phylum Cnidaria. Green hydra (Hydra viridissima Pallas) is endosymbiotic and brown hydra (Hydra oligactis Pallas) is a non-symbiotic species. Aluminium is one of the most abundant elements in the Earth's crust, but its effects upon living organisms have not been much studied until recently. The aim of this research was to explore the potential environmental effects of aluminium ions by studying aluminium-induced changes in two closely related hydra species, and to trace the extent damage caused. For the first time, a modified alkaline comet assay was used to study primary DNA damage in green and brown hydra cells. Aluminium toxicity triggered mortality, morphological, behavioral and DNA changes. DNA tail length and intensity changes were greater in brown than in green hydras, but behavioral responses to the presence of aluminium ions were observed more rapidly in green hydra. The toxicity also affected reproduction. Brown hydra was more susceptible to aluminium than green hydra, confirming the evolutionary advantage provided by symbiosis. Biomonitoring protocols using hydra and the comet assay could be developed to provide a valuable and rapid method for determining the quality of freshwater.

Since the effect of aluminium on living beings was little known until recently and it is possible to present a potential danger for freshwater organisms, the aim of this research was to explore its effect on green (Hydra viridissima) and brown (Hydra oligactis) hydra. Hydra is a simple metazoan organism that belongs to the Cnidaria phylum. It consists of ectoderm, mesoglea and gastroderm. Green hydra forms a symbiotic relationship with the endosymbiotic alga Chlorella, harboured inside the gastrodermal myoepithelial cells. Hydras were treated with four concentrations of aluminium sulphate (25, 250, 475, 500 mg/L) in laboratory conditions for three days and compared to the control. Only cellular layers of treated hydras contained the aluminium depositions: in green hydras depositions were found in gastroderm, including algae Chlorella, and in brown hydras they were found in ectoderm. We assume the appearance of these depositions to be the mechanism of cellular detoxication.

The effect of aluminum on the planarian Polycelis felina Daly. in laboratory conditions was studied. Planarians are simple freshwater invertebrates which belong to the phylum Platyhelminthes, class Turbellaria, order Tricladida. Planarians were treated with four concentrations of aluminium sulphate (25, 200, 600 and 1100 mg/L ; Kemika, Zagreb). Changes were observed on the fifth day after the treatment as well as on the fifth day of post-treatment recovery period. The results showed distinguished morphological changes on the planarian body as well as behavioral changes: various depigmentations, disordered locomotion, twisting of the body parts, hardly reacting to mechanical stimuli, also body contractions and mortality in the highest concenration. This work should also involve histomorphometrical analysis, showing if, how and where aluminum affected, and could be deposited in the planarian cells and tissue.

Aluminum is one of the most abundant chemical elements in the Earth's crust. The effect of aluminum on living beings was little known until recently. Since it is possible for the aluminum to be drained to surface and subterranean waters and to present the potential environmental danger for freshwater organisms and ecosystems, the aim of this research was to explore the effect of concentrated aluminum ions in aquatic environment and to observe the extent of damage it can do to brown and green hydra. Hydra is a member of the phylum Cnidaria, usually found in unpolluted freshwater. It has a simple, cylindrical body with an adhesive foot on one end and a mouth surrounded by six to eight tentacles on the other. It comprises two cellular layers separated by mesoglea. Green hydra forms a symbiotic relationship with algae of Chlorella genus. Hydras regenerate at a tremendous rate. Comparative static toxicity test was performed using the individuals of brown (Hydra oligactis Pallas, 1766 ; sort S1M-K1) and green (Hydra viridissima Pallas, 1766 ; sort S1J-J1) hydra. They were treated with seven concentrations of aqueous solution of aluminum sulphate (25, 50, 80, 100, 150, 200 and 250 mg/L ; Kemika, Croatia) in laboratory conditions (22.80 C) in subacute exposure for five days and compared to the control groups of organisms. After the exposure, animals were put in clean aquarium water, recovering for ten days. For cytological and histological analysis hydras were fixed in Bouine fixative after the period of recovery. Preparations were differentially stained with 0.2 % acid eriochrome cyanine, and 0.1 % toluidine blue pH 4.4 and hämalaun-eosine. Using a sample of 200 cells, we determined the percentage containing aluminum depositions and measured the surface area of those depositions in comparison to the area of cells containing them. Histomorphological and morphometrical analysis was performed by using Lucia G DXM1200 version 4.81 software. Micrographs were made by using Reichert and Nikon Eclipse E600 microscopes, Pentax and Nikon DXM1200 cameras. Higher concentrations of aluminum triggered greater morphological and cytological damages. Both mucous secretion of the foot region and tentacle reduction were noted. Cytotoxic effects of higher concentrations yielded a greater number of aluminum containing cells and larger deposition surface in the cells. Aluminum depositions were found in ectodermal cells only, in the form of single and multiple (2-4 separate) depositions and clusters. No significant differences concerning aluminum depositions were observed between brown and green hydra. Control animals did not show any changes. Aluminum was proved to be a potentially hazardous freshwater pollutant and showed a strong deleterious effect on hydras. In spite of its potent deleterious impact, hydras regenerated nearly completely as only aluminum deposits remained. Since depositions were found in ectodermal layer only, we suggest that mesoglea could present an aluminum buffer in itself. This was the first time green and brown hydras were treated with aluminum ions as well as the first time they were stained with eriochrome cyanine, using an appropriately modified technique. The results of this experiment provided many new interesting aspects for further research on evolutionary and ecotoxicological issues of hydra symbiosis, including TEM and SEM.

The effect of norflurazon on symbiotic green hydra (Hydra viridissima Pallas, 1766/strain S1J-J1), and asymbiotic brown hydra (Hydra oligactis Pallas, 1766/strain S1M-K1) and green alga Chlorella kessleri Fott et Novak. [K&H, 1992] was researched. Norflurazon is a selective translocational herbicide that causes &laquo ; bleaching effect&raquo ; upon the newly developed chloroplasts, resulting in the decrease of the viability of the organism. Green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells it contains individuals of Chlorella sp. In ecotoxicological research, this was the first example of such a series of test-organisms, which share the complex evolutionary and symbiotic relationships. The organisms were treated under both high- and low-light conditions for 21 days with five concentrations of aqueous solution of norflurazon (2x10-4, 2x10-5, 2x10-6, 2x10-7 and 2x10-8 mol/L ; SAN 9789, Sandoz Ltd., Basel, Switzerland) in the laboratory conditions. Conventional histological and TEM methods were used. Higher concentrations and high-light conditions caused increased mortality, significant morphological, cytological, histological and ultrastructural changes, changes in behavior, locomotion and asexual reproduction in all the test-organisms. Damages to aposymbiotic organisms were much more expressed than in the symbiotic organisms. In low concentrations of norflurazon, the similarity to the control and hormestic effect upon the algal growth was manifested. Well defined green hydra endosymbiosis was disturbed. Of special significance were antichloroplastal and antimitochondrial effects, as well as damages to almost all the organelles of the organisms. Newly developed buds in green hydra were not bleached, while the bleaching effect was present only in the algae out of the symbiosis. Symbiotic relationship in green hydra was confirmed firm and stable. The final result of the disturbed symbiosis between hydra and alga was the reassembling of the endosymbiosis in particular individuals that survived, as well as their partial or full recovery. By analyzing the green hydra symbiosis in itself, symbiotic Chlorella performed as the stronger link in this symbiotic relationship. Endosymbiotic alga was less affected by intoxication than green hydra itself. Hydra suffered from overall greater percentage of damages than the alga. It can be concluded that symbiosis provides greater survival potential in a toxic environments and represents an evolutionary advantage. It appears that complex symbiotic relationship as a result of pre-adaptations blocks the bleaching effect in green hydra. When it came to survival, symbiotic alga seemed to present the stronger component of the green hydra symbiosis. Instead of the usual cooperation between two endosymbiotic partners, in a sense alga performed a cheating mechanism in a toxic environment.

U radu je opisana evolucija uha i slušnog aparata te uloga uha u osjećanju gravitacije i održavanju ravnoteže

The effect of norflurazon on green and brown hydra was studied on ecotoxicological and evolutionary level. The aim was to compare changes between two closely related hydra species, to trace differences of the effect of herbicide upon them and to draw out the conclusion about sustainability and advantage of hydra symbiosis. Green hydra (Hydra virdissima Pallas, 1776) is a typical example of symbiosis (1). In its gastrodermal myoepithelial cells it contains the individuals of unicellular green alga Chlorella (Fig. 1, (3)). Brown hydra (Hydra oligactis Pallas, 1766) is a nonsymbiotic hydra. Herbicide norflurazon (nf) causes chloroplastal bleaching. Individuals of green (sort S1J-J1) and brown (sort S1M-K1) hydra were treated with five concentrations of aqueous solution of norflurazon (2x10^-4, 2x10^-5, 2x10^-6, 2x10^-7 and 2x10^-8 mol/L ; SAN 9789, Sandoz, Switzerland) in the laboratory conditions (21o C, photoperiod 10 h daylight and 14 h dark) in subacute exposure for 21 days. They were compared to the control groups of organisms. For morphological analysis binocular light microscope Carl Zeiss, Jena was used. For cyto-histological analysis hydras were fixed in Bouine fixative 24, 48 and 72 h after the beginning of the experiment. Preparations were dyed with 0.1% toluidine blue pH 4.4 and hämalaun-eosine (he). Micrographs were made by using Reichert and Nikon Eclipse E600 microscopes, Pentax and Nikon DXM1200 cameras. Nf toxicity resulted in numerous structural changes, showing the strong deleterious effect upon both hydra species, damaging almost all their structures. Depending on the concentration, nf affected viability and caused mortality, significant morphological, cyto-histological changes, changes in locomotion, behavior and asexual reproduction. In all parameters brown hydra showed higher nf susceptibility. Most prominent damages to the animal cells were apocrine secretion around the animal body and damages to ectodermal and gastrodermal myoepithelial cells. Glycocalyx was partially missing. Gastral cavity was filled with different cellular components. I- and zymogene cells were in the process of dedifferentiation to other cell types and migration across the body axes (Figs. 2-4). Endosymbiotic algae fell out of their host cells. They were reduced in number, changed in shape, size and color and chloroplastal degradation was present. Newly developed buds were not bleached. The final result of the disturbed symbiosis of green hydra was the reassembling of the endosymbiosis in particular individuals that survived, as well as their partial or full recovery. Since there was no bleaching noticed, it seemed that the symbiotic interactions, which are the result of symbiont's preadaptations, block the chloroplastal bleaching in green hydra. Therefore it could be concluded that endosymbiotic Chlorella from green hydra could perform some level of resistance to toxicants (2). This result could support the ever-stronger idea that Chlorella from green hydra represents a separate species of algae (4). Also, as brown hydra showed higher level of nf susceptibility than green hydra, symbiosis confirmed to present the evolutionary advantage. Research in this field contributes to resolving and better understanding of the complex problematic of symbiosis in fundamental biological science.

Aluminum (Al) is one of the most abundant chemical elements in the Earth's crust. Yet, the effect of Al on living beings was little known until recently. This was partly because Al becomes soluble in water with increasing acidity only, and it was essentially harmless until acid rains first appeared. Because it is possible for the Al to be a major constituent of surface and subterranean waters, thereby presenting a potential environmental danger for freshwater organisms, the aim of this research was to compare the changes between two closely related hydra species. Specifically, we sought to trace differences and the extent of damage of the effect of Al upon green and brown hydra. Usually found in unpolluted freshwater, hydra is in the the phylum Cnidaria. It has a simple, cylindrical body with an adhesive foot on one end and a mouth surrounded by five to eight tentacles on the other. It comprises two cellular layers separated by mesoglea. Green hydra forms a symbiotic relationship with Chlorella algae. Hydra has a tremendous rate of regeneration. A comparative static toxicity test was performed using the individuals of green (Hydra viridissima Pallas, 1766 ; strain S1J-J1) and brown (Hydra oligactis Pallas, 1766 ; strain S1M-K1) hydra. They were treated with 9 concentrations of aqueous solution of aluminum sulphate (25, 50, 80, 100, 150, 200, 250, 350 and 500 mg/L ; Kemika, Croatia) in laboratory conditions (22.8oC) in subacute exposure for three days and compared to the control groups of organisms. After the exposure, the animals were put in clean aquarium water, and left recovering for 14 days. Al toxicity triggered numerous morphological and behavioral changes, showing the strong deleterious effect on both hydra species. Brown hydra showed higher Al susceptibility in all parameters. Higher concentrations triggered more prominent changes: tentacle reduction, incoherent and sluggish reactions to mechanical stimuli, migration towards water surface and apocrine mucous secretion of ectodermal layer. It strongly induced a hormestic effect in the asexual reproduction of green hydra and even induced sexual reproduction. Mortality was noted only in the highest concentration. An interesting hypothesis was confirmed wherein the brown hydra was able to regenerate itself to its original height by elongating its epithelial cells. This ability in the green hydra is yet to be examined. Control animals did not show any changes. As brown hydra showed higher level of Al susceptibility than green hydra, symbiosis seemed to present an evolutionary advantage. Higher water acidity levels greatly increased the overall toxicity of aluminum to these species. Greater Al concentrations brought harmful impact to both species. However, cooperative ad symbiotic interactions between the green hydra and the green algae give the symbiont stronger and better resistance compared to the brown hydra. This hydra bioassay was shown to be a useful tool for quick corroboration of the expected results.

Symbiotic associations are of wide significance in evolution, providing biological advantages and biodiversity. Mutualism is a form of symbiosis where both partners benefit from the relationship, while in parasitism only one partner benefits on the count of the other. It is presumed that symbiotic relationships arose from parasitic attempts. In this work an example of a rare process of evolutionary reversal was described on green hydra symbiosis. Green hydra (Hydra virdissima Pallas, 1776) is a typical example of symbiosis. In its gastrodermal myoepithelial cells it contains the individuals of unicellular green alga Chlorella, each alga surrounded by perialgal space and placed in a separate vacuole called symbiosome. Triggers for observing the described changes were herbicide norflurazon (nf) and antibiotics chloramphenicol (cfc), cinoxacin (cin) and ciprofloxacin (cip). Individuals of green hydra (sort S1J-J1) were treated with aqueous solutions of nf (2x10-4, 2x10-5, 2x10-6, 2x10-7 and 2x10-8 mol/L ; SAN 9789, Sandoz, Switzerland), cfc (100 and 250 mg/L ; Pliva, Croatia), cin (62.5 and 117.6 mg/L ; Lilly Deutschland, Germany) and cip (1 and 5 mg/L ; Bayer AG, Germany) in the laboratory conditions (21o C, photoperiod 10 hs daylight and 14 hs dark) in subacute exposure for 3-21 days. They were compared to the control groups of organisms. For TEM, standard preparation methods were used. Micrographs were made by using Zeiss EM10A and FEI Morgagni 269D microscopes. Toxicity resulted in numerous ultrastructural changes, showing a strong deleterious effect upon both hydra and alga FIG. 1. Hydra suffered from overall greater extent of damages than alga did. Well-defined green hydra endosymbiotic relationship was disturbed. Alga showed interesting and independent protective mechanisms by widening of perialgal space to the final extent, which resulted in symbiosome reduction or loss FIG. 1. In this way alga regulated itself a more viable environment than the host. Formations of two to three algae contained inside a single symbiosome in which algae “ joined forces” for their protection were noticed as well. By these mechanisms algae showed a certain degree of independence from their host. When it came up to survival, alga seemed to be the stronger component in this symbiotic relationship. It seemed that during millions of years of coevolution endosymbiotic alga gained the specific preadaptations by which it defended itself from environmentally harmful effects better than the host green hydra itself. The following question arose: could we discuss the possibility of alga to transform the green hydra endosymbiotic relationship from mutualism to parasitism, for at least a very short interval where it survived longer and better than its host. The lateral gene transfer from alga to hydra is well supposed. But, could it be that a part of genetic material of hydra became dependent on alga? It came to re-questioning of the host-symbiont relationship, which opens many research possibilities. The results supported the ever-stronger idea that Chlorella from green hydra represents a separate species of algae.

Fumonisins are a family of mycotoxins produced by Fusarium sp. (mostly F. moniliforme and F. proliferatum). Exposure to fumonisins is associated with a variety of adverse health effects in domestic and experimental animals. Among more than 10 types of fumonisins, the most prevalent and the most toxic is fumonisin B1 (FB1). Apoptosis is a programmed cell death characterized by a cytoplasmic shrinkage and condensation of chromatin in nucleus. The aim of this study was to assess whether low doses of FB1 induce apoptosis in rat liver at different lag time after treatment. Adult, male Wister rats (6 per group) were given single oral dose of FB1 (5, 50, and 500 μ g/kg b.w. dissolved in sterile saline) and sacrificed 4, 24, and 48 hours afterwards. Control animals were given sterile saline or Fe-NTA. Whole liver was fixed in formalin, routinely processed, and embedded with paraplast. Sections of 4 &micro ; ; ; m were stained with hematoxyllin and eosin (HE). Number of hepatocytes undergoing apoptosis was determined by counting 500 cells (computer software Lucia G 4.81) in 25-50 randomly selected microscopic fields of vision (microscope Nikon Eclipse E600). Data were presented as mean with standard deviation, median, minimum and maximum values, and geometrical mean. Possible differences in treatment and control groups were evaluated using t-test. Probability values of P

Although aluminum is one of the most abundant chemical elements in the Earth's crust, its effect on living beings was little known until recently. Since the effect of aluminum ions upon green and brown hydras has not been studied yet, with this technical note we showed how aluminum depositions in symbiotic and asymbiotic hydras could be detected, observed and analyzed. Differential eriochrome cyanine staining on hydras has been performed for the first time, using an appropriately modified technique for detecting the aluminum depositions in hydras. This hydra bioassay was shown to be a useful and exact tool for detection of expected results e.g. aluminum depositions in hydra cells.

The aim of this study was to characterize and quantify hemocyte types in Indian stick insect (Carausius morosus), American cockroach (Periplaneta americana), Deathshead cockroach (Bleberus craniifer) and Cave cockroach (Archimandrita tessellata). Total (THC) and differential (DHC) hemocyte counts were performed and the results were compared. Insects are by far the largest and the most heterogeneous group of organisms on Earth that dominate land found in freshwater and sea as well. It is estimated that there are at least 800 000 species of insects on Earth today. Although insects have been studied for years, their circulatory system has remained unexplored to great extent. The insect body fluid, hemolymph, circulates freely around the internal organs (2). It consists of hemocytes and the fluid. Hemocytes, insect blood cells, play an important role in the insect immune system (3). Wider knowledge on hemocytes would contribute to a better understanding of the insect immune system (1). Insects were kept on room temperature (24°C) and fed with ivy leaves. They were sprinkled with water once a day. To collect hemolymph, insects were put in hot water (60°C) for two minutes, to prevent blood coagulation. Hemolymph was transferred on a glass coverslip with a micropipette. Hemocytes were fixed in methanol for 15 minutes and dyed with Giemsa stain (G) for three minutes. THC was performed by standard procedure in Bürker-Türk chambers on five individuals of each species. For DHC a hundred hemocyte cells per individual were analyzed, five individuals per species. Both THC and DHC were made by using Olympus CX41 light microscope. Preparations were photographed by using Olympus E-1 digital camera and "QuickPhotoPRO 2.0" computer program. THC in species B. craniifer varied between 63-90 000 hemocytes/&micro ; L of hemolymph, while DHC showed that there were present five types of hemocytes: plasmatocytes (34-52 %), spherulocytes (35-44 %), prohemocytes (1-3 %), granulocytes (7-15 %), coagulocytes (1-6 %). THC in species P. americana varied between 70-120 000 hemocytes/&micro ; L of hemolymph. DHC in this species showed three types of hemocytes: plasmatocytes (86-91 %), prohemocytes (6-11 %), coagulocytes (3-5 %). THC in A. tessellata varied between 33-60 000 blood cells/&micro ; L of hemolymph. DHC showed five types of blood cells: spherulocytes (45-56 %), plasmatocytes (17-29 %), granulocytes (10-15 %), prohemocytes (3-8 %), coagulocytes (3-8 %). In C. morosus THC varied between 11-17 000 hemocytes/&micro ; L of blood. DHC showed 4 types of cells: plasmatocytes (66-76 %), prohemocytes (10-18 %), granulocytes (8-12 %) and coagulocytes (4-9 %). Although the three out of four species of studied insects belong to the same order (Dyctioptera), two of which belong to the same suborder (Blaberidae), the differences in THC between the species were extremely high. The lowest THC of all studied species has C. morosus. Although B. craniifer and A. tessellata belong to the same suborder, their THC was not alike. The biggest differences were visible between P. americana and other cockroaches. We suppose that those differences occurred because P. americana is not a tropical species as the other, and their habitats differ. DHC varied from species to species. Tropical species of cockroaches (B. craniifer, A. tessellata) had the most similar DHC. They had the highest concentration of spherulocytes, which were not found in other species. All the species contained prohemocytes, plasmatocytes and coagulocytes. P. americana did not contain granulocytes and spherulocytes, while C. morosus did not contain spherulocytes. Plasmatocytes and spherulocytes were highly concentrated in the hemolymph. All the species had a relatively low concentration of coagulocytes in their blood. Wider knowledge of insect THC and DHC could be the key to understanding their defense system, since hemocytes play an important role in phagocytosis and blood coagulation.

The effect of norflurazon on endosymbiotic Chlorella vulgaris Beij. [K&H, 1992] from green hydra (Hydra viridissima Pallas, 1766) and asymbiotic Chlorella kessleri Fott et Novak. [K&H, 1992] was researched on evolutionary and ecotoxicological level. With this work we tried to obtain the information about difference between high-related algae in symbiosis and out of it and to observe the differences of the effect of toxicant upon them. Norflurazon causes the 'bleaching effect' upon the newly developed chloroplasts. Organisms were treated with aqueous solutions of norflurazon (2x10-5, 2x10-6, 2x10-7 mol/L) in glass dishes, and in tubes on deep stock agar for 21 days in the laboratory conditions. Cytological and ultrastructural changes were observed with binocular light microscope and TEM. Of special significance were the antichloroplastal effect and damages to the thylakoidal system, which were increased with the higher concentration of toxicant. Newly developed buds in symbiotic green hydra were not bleached, and therefore neither were endosymbiotic algae. The 'bleaching effect' was present only in algae out of symbiosis, with deleterious effect upon algal chloroplasts. This could suggest that endosymbiotic alga could have some level of resistance to toxicants or could even support the idea that Chlorella from green hydra could present the separate species of algae.

Mechanisms of green hydra symbiosis were described. Herbicide norflurazon was chosen as a "trigger" for observing of these mechanisms. Green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells it contains the individuals of Chlorella vulgaris Beij. (K&H, 1992) species (so far data). Ultrastructural changes were observed by means of TEM. Newly observed, described and estblished mechanisms in green hydra symbiosis were widening of the perailgal space, missing symbisomes and joining of the existing perialgal spaces. Also, on the basis of these newly described mechanisms, recovery of green hydra after the intoxication period was explained. The final result of the disturbed symbiosis between hydra and alga was the reassembling of the endosymbiosis in particular individuals that survived.

Reaction of OH radical with fluorinated and chlorinated benzene is considered a model reaction for the atmospheric degradation of halogenated aromatic toxic pollutants (dioxins, biphenils...). The addition of OH radical to fluorinated and chlorinated benzene has been investigated by using Moller-Plesset ab initio (MP2/6-31+G(d, p)) and B3LYP density functional calculations (B3LYP/6-31G(d, p)). The geometries of prereaction complexes, transition states, and products are determined for addition on all possible positions on the aromatic ring. Arrhenius rate constants are calculated for these reactions.