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Process of endosymbiosis is one of promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. Its gastrodermal myoepithelial cells harbor the individuals of unicellular photoautotrophic algae. We have established prosperous laboratory cultures of endosymbiotic green algae isolated from green hydra strains. Hydra strains were from 6 different geographical localities: 4 from Croatia, 1 from Israel and 1 from Germany. Endosymbiotic green algae were successfully isolated using microbiological methods. Isolated endosymbiotic algae were characterized employing cytological morphometric parameters, enzyme activity and isoenzyme pattern analysis (catalase, peroxidase and esterases). Cells of endosymbiotic algae were characterized by morphometric measurements: diameter, perimeter and area. Algal HV and M9 strains were separated from strains T and BV. The results obtained by isoenzyme pattern analysis suggest a difference between Croatian and European algal endosymbionts, indicating biological diversity among algal symbionts isolated from green hydras. Five isoenzymes of catalase and five isoenzymes of peroxidase were resolved by PAGE electrophoresis. Isoenzyme K1 appeared only in endosymbiotic algae M9 from Israel and isoenzyme K3 only in T strain. Isoenzyme P1 was observed only in endosymbiont HV while isoenzyme P5 in both endosymbionts HV and M9. Isoenzymes P3 and P4 were visible only in algal strains BV and T from Croatia.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

The effect of chloramphenicol on the symbiosis between alga and hydra.

Metoda mikrokozmosa koristi se u proučavanju populacijske dinamike predatora i plijena i istraživanju prehrambenih lanaca, što daje mogućnost za modeliranje i rekonstrukciju odnosa u populacijama i biocenozama u prirodi. Cilj ovog rada je istraživanje interspecijskih i intraspecijskih odnosa kompeticije, predacije i simbioze u slatkovodnom mikrokozmosu, između zelene hidre, mnogooke i šiljoglave puzavice, velike vodenbuhe i izolirane endosimbiotske alge. Cilj je i istražiti zastupljenost izvannastavnih aktivnosti iz područja prirodoslovlja među učenicima osnovnih škola i atraktivnost sadržaja koji bi se mogao ponuditi u okviru izvannastavnih aktivnosti u osnovnoj školi te atraktivnost navedenog pokusa u osnovnoj školi. Svaki pokus izveden je u pet replika pri 25 i 13,5 °C, sa sitim i s gladnim životinjama, uz postavljanje kontrola. Pokazano je da je zelena hidra izuzetno zanimljiv i jak kompetitor i predator u slatkovodnom mikrokozmosu te da alge stvaraju mrežu, sudjelujući u predaciji, dok virnjaci predacijom stvaraju kamuflažnu obojenost. Podaci o zastupljenosti izvannastavnih aktivnosti iz područja prirodoslovlja i atraktivnosti sadržaja koji bi se mogao ponuditi u okviru izvannastavnih aktivnosti u osnovnoj školi prikupljeni su metodom anonimnog anketiranja. Zastupljenost izvannastavnih aktivnosti iz prirodoslovnog područja među anketiranim je učenicima vrlo niska, a atraktivni su im sadržaji koji obuhvaćaju praktični rad iz područja biologije, pa provedeno istraživanje može biti dobar temelj za izvannastavne aktivnosti u osnovnoj školi. The microcosm method is used in the study of population dynamics of predation and prey and in the research of of food chains, which provides an opportunity for modeling and reconstruction of relationships in populations and biocenoses in nature. The aim of this paper is to investigate the interspecies and intraspecies relationships of competition, predation, and symbiosis in freshwater microcosms, between green hydra, freshwater turbellarians, large water fleas, and isolated endosymbiotic algae. The aim is also to study the share of extracurricular activities in the field of science among primary school students regarding the attractive content that can be offered within extracurricular activities in primary school and the attractiveness of attending an experiment. Experiment was performed in five replicates at two different temperatures (25 °C with a photoperiod of 8 h day/16 h night and at 13, 5 °C in the dark) and with fed and hungry animals, including control replicates. It has been shown that the hydra is the most interesting and most important competitor and predator and that the algae create a web by participating in the predation, while the turbellarians create a camouflage coloration as a result of predation. The method of anonymous survey results show that the prevalence of extracurricular activities in the field of science among the surveyed students is very low, and that they are attracted to the contents that include practical work in biology, especially those using a microscope, so the proven research can be a good basis for extracurricular activities in primary school.

Postoje dokumentirana istraživanja o postojanju tumora na tijelu hidri, a ovo je prvo istraživanje gdje su jedinke hidre izlagane tretmanu UVB-zračenjem i/ili norflurazonu s namjerom da se izazovu tumori. Jedinke zelene hidre (Hydra viridissima Pallas, 1766) i jedinke smeđe hidre (Hydra oligactis Pallas, 1766) ili obje vrste zajedno izlagane su norflurazonu koncentracija 2x10-7 mol/L ili 2x10-6 mol/L. Dio hidri izložen je norflurazonu i zračen UVB-zračenjem valne dužine 254 nm 2 minute. Drugi dio pokusa je rađen tako da su hidrama dodane suspenzije slobodnoživuće alge Chlorella vulgaris (CV) i endosimbiotske alge Mychonastes homosphaera (CZ10) ili obje alge zajedno. Hidre s dodatkom algi su izlagane norflurazonu i zračene na isti način kao u prvom dijelu pokusa. Pratile su se morfološke promjene hidri: migracija, odgovor na mehanički podražaj, oblik tijela, oštećenje lovki, nespolno (pupanje) i spolno razmnožavanje te smrtnost. U eksperimentalnim posudicama s dodatkom algi pratile su se još boja otopine i prisustvo nakupina algi. Migracija hidri, promjena oblika tijela pretežno iz relaksiranog u kontrahirani oblik i spolno razmnožavanje bili su prvi znakovi izbjegavanja štetnog učinka UVB-zračenja ili norflurazona. Histološke promjene pratile su se svjetlosnim mikroskopom i brojale su se stanice ektoderma, gastroderma, intersticijske stanice, zimogene stanice i žarne stanice, a mjerena je i debljina mezogleje. Ugibale su samo zračene jedinke zelene hidre. Pri višoj koncentraciji norflurazona i u svim zračenim hidrama uočena su velika oštećenja u strukturi tkiva hidri. Zračenje je prouzročilo izrasline na lovkama i na tijelu hidre ili specifične rašljaste lovke The occurence of tumors on the body of hydra has already been reported in researches but this is the first study in which the individuals of symbiotic green hydra and nonsymbiotic brown hydra were exposed to UVB-radiation and /or norflurazon in order to induce tumors. The individuals of green hydra (Hydra viridissima Pallas, 1766) and brown hydra (Hydra oligactis Pallas, 1766), either separately or both species together, were treated with norflurazon in concentrations of 2x10-7 mol/L or 2x10-6 mol/L. A group of hydra was treated with norflurazon and exposed to UVB-radiation with a wavelenght of 254 nm for 2 minutes. The second part of the experiment was performed by adding suspensions of free-living algae Chlorella vulgaris (CV) or endosimbiotic Mychonastes homosphaera (CZ10) and both algae together. The group of hydra with algae suspension was treated with norflurazon and exposed to UVB-radiation in the same way as in the first part of the experiment. The following morphological changes of hydra were observed: migration, response to mechanical stimuli, body shape, damage to tentacles, asexual (budding) and sexual reproduction, and mortality. The experimental dishes with algae suspension were monitored for the change of solution color and the presence of algae blobs. Migration, change of body shape from relaxed to predominantly contracted, and sexual reproduction were identified as the first evasion signs of the deleterious effects of UVB-radiation and norflurazon. Histological changes were detected by light microscope, while ectoderm, gastroderm, interstitial, zymogen and cnidocyte cells were counted. The thickness of mesoglea was measured. Mortality was present in the individuals of green hydra that were exposed to UVB-radiation. Severe damage to the structure of hydra tissue was noted in hydra treated with high concentrations of norflurazon and in all hydra exposed to UVB-radiation. Radiation caused tissue growths on both tentacles and body of hydra as well as specific fork-shaped tentacles in hydra.

Simbiotske asocijacije imaju široki značaj u evoluciji i bioraznolikosti. Proces endosimbioze jedan je od pokretača specijacije. Zelena hidra (Hydra viridissima Pallas, 1766) tipičan je primjer endosimbioze, jer u svojim gastrodermalnim mioepitelnim stanicama sadrži jednostanične fotoautotrofne alge. Endosimbiotske alge izolirane iz zelenih hidri sakupljenih s četiri različite lokacije u Hrvatskoj, te iz Izraela i iz Njemačke identificirane su koristeći pet molekularno filogenetičkih biljega (geni za 18S rRNA, za ITS regiju, za 16S rRNA, za rbcL i za citokrom oksidazu I) pomoću metode najveće vjerojatnosti. Istražen je filogenetički položaj endosimbiotskih algi izoliranih iz zelenih hidri unutar zelenih algi Chlorophyta. Molekularno filogenetičkom analizom utvrđeno je da simbiotske alge u zelenoj hidri pripadaju različitim rodovima i vrstama jednostaničnih zelenih algi iz razreda Trebouxiophyceae i Chlorophyceae. Simbiotske alge izolirane iz zelenih hidri s hrvatskih lokaliteta pokazuju polifiletičko podrijetlo nastanka na različitim geografskim lokacijama. Izolirane endosimbiotske alge karakterizirane su pomoću svjetlosne te pretražne i transmisijske elektronske mikroskopije, prema citološko-morfometrijskim parametrima te prema aktivnosti i sastavu enzima katalaze, peroksidaze i esteraza. Rezultati dobiveni analizom sastava izoenzima upućuju na razliku između algalnih endosimbionata s hrvatskih lokaliteta i algalnih endosimbionata iz Europe, ukazujući na biološku raznolikost među algalnim simbiontima. Symbiotic associations are of a broad significance in evolution and biodiversity. The process of endosymbiosis is one of promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells harbors the individuals of unicellular photoautotrophic algae. We reconstructed the phylogeny of algal endosymbionts isolated from green hydra strains collected from four different geographical sites within Croatia and one from Germany and one from Israel. Nuclear (18S rRNA, the ITS region), chloroplast (16S rRNA, rbcL) and mitochondrial (COI) markers for maximum likelihood phylogenetic analyses were used. We focused on investigating the positions of these algal endosymbiotic strains within the chlorophyte lineage. Molecular analyses established that different genera and species of unicellular green algae from class Trebouxiophyceae and Chlorophyceae are present as endosymbionts in green hydra, showing that are of polyphyletic origin. Our results indicate that the intracellular algal endosymbionts of green Hydra have become established several times independently in evolution. Isolated endosymbiotic algae were characterized by light microscopy and scanning and transmission electron microscopy, according to cytological morphometric parameters and by activity and isoenzyme analysis of catalase, peroxidase and esterases. The results obtained by isoenzyme analysis suggest a difference between Croatian and European algal endosymbionts, indicating biological diversity among algal symbionts.

Željezo je jedan od najraširenijih metala u Zemljinoj kori. Hidra je slatkovodna životinja koljena Cnidaria. Toksični učinak željeza na različite organizme bio je već predmet istraživanja, ali ovo je prvo dokumentirano istraživanje o njegovu učinku na hidre. Zelena hidra sadrži u svojim gastrodermalnim mioepitelnim stanicama simbiotsku algu. Jedinke simbiotske zelene hidre bile su tretirane sa šest koncentracija vodene otopine željeznog dekstrana, dok je nesimbiotska smeđa hidra bila tretirana s 14 koncentracija vodene otopine željeznog dekstrana tijekom tri dana. Morfološke promjene su praćene pomoću stereolupe i to migracija jedinki u eksperimentalnoj posudici, promjena oblika tijela, reakcija na mehanički podražaj, odsutnost lovki, lučenje sluzi i nespolno razmnožavanje. Migracija hidri je prvi način izbjegavanja štetnog učinka (mikro)okoliša, nakon čega je uslijedila promjena oblika tijela. Histološke promjene praćene su pomoću svjetlosnog mikroskopa. Morfometrijska istraživanja obuhvaćala su mjerenja površina ektoderma i gastroderma te debljine mezogleje. Depozicije željeza u tkivu hidri su bile vidljive su kao plavo obojenje unutar stanica. Visoke koncentracije željeza uzrokovale su morfološke i histološke promjene i letalni učinak. Zelena hidra je pokazala veću otpornost u preživljavanju od smeđe. Simbioza se pokazala kao evolucijska prednost. Iron is among the most 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 investigated 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 hydra was treated with 14 concentrations of aqueous solution of iron dextran for three days. Morphological changes migration of individuals in experimental dish, changes of body shape, reaction to mechanical stimuli, tentacle reduction, mucous secretion and asexual reproduction were monitored with stereolupe. Migration of hydras was the first step in evading deleterious microenvironmental conditions, followed by changing its body shape. Histological changes were observed with light microscope. Morphometric analysis included measuring of endoderm and gastroderm surface areas and mesoglea thickness. Iron depositions in hydra tissue were observed as blue staining within cells. High concentrations of iron caused morphological and histological changes and mortality. Green hydra showed a higher level of resistance in surviving than brown hydra. Symbiosis was pointed out as an evolutionary advantage.

Simbioza u praživotinja uglavnom predstavlja bliski mutualistički suživot između praživotinje i jednostaničnih simbionata (bakterija, cijanobakterija i/ili jednostaničnih alga) ili praživotinja i višestaničnog organizma (preživač, niži termit ili žohar koji se hrani drvetom, biljka). Dobrobit simbioze za praživotinjskog domaćina može biti: dodatna opskrba hranjivim tvarima od jednostanične alge ili cijanobakterije, zaštita od predatora i opskrba kisikom. Dobrobit simbioze za simbionte može biti: zaštita od predatora i opskrba hranjivim tvarima. Jedna praživotinja može imati i nekoliko stotina jednostaničnih simbionata. Simbioza može biti obligatna ili fakultativna, endosimbioza ili ektosimbioza. Simbioza je uobičajena u trepetljikaša i sluzavaca. Najbolje istražena simbioza je ona između trepetljikaša roda Paramecium i jednostaničnih algi roda Chlorella. Ovaj rad je kratka prezentacija uobičajenih primjera simbioze u praživotinja. Govori o nužnosti prilagodbe simbionta-domaćina i simbionta-gosta za uspostavljanje i održavanje simbioze. Važnost simbioze je u tome što omogućuje ovim organizmima zauzimanje novog mjesta u ekološkoj niši, koje je nedostupno drugim organizmima. Simbioza u praživotinja u prirodi je vrlo česta i raznolika, ali još uvijek nedovoljno istražena. Symbiosis in protozoa mostly represents a close mutualistic association between a protozoan and unicellular symbionts (bacteria, cyanobacteria or/and unicellular algae) or protozoans and a multicellular organism (ruminants, lower termites, wood-eating cockroaches, plants). Benefits for a protozoan host can be: extra food supply from intracellular algae or cyanobacteria, protection from predation and oxygen supply. Benefits for symbionts can be: protection from predation and nutrition supply. One protozoan can bear a couple of hundreds unicellular symbionts. Symbiosis can be obligate or facultative, endosymbiosis or ectosymbiosis. It is very common in ciliate and sarcodine protozoans. The best studied symbioses are those between a ciliate protozoan of genus Paramecium and symbiotic unicellular algae of genus Chlorella. This work presents a short overview of the most common examples of symbiosis in protozoa. It shows the necessity of adaptations of symbionts and hosts for formation and maintainance of the symbiosis. Symbiosis is very important because it allows these organisms to take new place in ecological niche which is out of reach to other organisms. Symbiosis in protozoa in nature is very common and shows a great diversity, but is still not adequately investigated.