ISSN 0378-2697, Volume 284, Combined 1-2

This article was published in the above mentioned Springer issue. The material, including all portions thereof, is protected by copyright; all rights are held exclusively by Springer Science + Business Media. The material is for personal use only; commercial use is not permitted. Unauthorized reproduction, transfer and/or use may be a violation of criminal as well as civil law. Plant Syst Evol (2010) 284:33–39 Author's personal copy DOI 10.1007/s00606-009-0235-z

ORIGINAL ARTICLE

Morphological features and isoenzyme characterization of endosymbiotic algae from green hydra

Goran Kovacˇevic´ • Sandra Radic´ • Biserka Jelencˇic´ • Mirjana Kalafatic´ • Hrvoje Posilovic´ • Branka Pevalek-Kozlina

Received: 10 April 2009 / Accepted: 9 October 2009 / Published online: 27 October 2009 Ó Springer-Verlag 2009

Abstract Symbiotic associations are of a wide signifi- Keywords Endosymbiotic alga Green hydra cance in evolution and biodiversity. Green hydra is a typ- Morphometry Isoenzymes ical example of endosymbiosis. In its gastrodermal myoepithelial cells, it harbors individuals of unicellular green algae. Morphological characteristics of isolated algae Introduction determined by light and electron microscopy are presented. Cytological morphometric parameters (cell area, cell Hydra (Cnidaria, Hydrozoa) is an aquatic cosmopolitan radius, chloroplast area) of isolated algae from green hydra invertebrate (Holstein and Emschermann 1995) that rep- (Cx), as well as from reference species Chlorella kessleri resents a very fine object for experimental purposes (Ck) and Chlorella vulgaris (Cv), revealed similarity (Arkhipchuk et al. 2005; Beach and Pascoe 1998; Kalafatic´ between the isolated endosymbiont and C. kessleri. Iso- and Kopjar 1995; Kovacˇevic´ et al. 2001). enzyme patterns of esterase (EST), peroxidase (POX), and Green hydra (Hydra viridissima Pallas, 1766) is an catalase (CAT) were used for the investigation of genetic endosymbiotic species that harbors individuals of unicel- variability in endosymbiotic algae isolated from green lular green algae in its gastrodermal myoepithelial cells hydra. Out of 14 EST isoenzymes observed in Cx species, (Burnett 1973; Douglas and Smith 1984). In one cell up 9 were expressed in the Cx sample. Results of the EST to 20 algae can be found (Dunn 1987; Holstein and isoenzyme analysis indicated a higher degree of similarity Emschermann 1995) with each alga surrounded by a vac- between Cx and Cv than between Cx and Ck. Due to much uolar membrane, forming a symbiosome (O’Brien 1982; higher heterogeneity, EST isoenzymes seem to be more Reisser and Wiessner 1984). suitable genetic markers for identification of different Individuals and species that do not establish an endo- Chlorella species than CAT and POX isoenzymes. Results symbiotic relationship are called asymbiotic or nonsymbi- obtained suggest that symbiogenesis in green hydra has otic, while organisms that can form endosymbiotic probably not been terminated yet. relationships in certain periods and then again live freely as separate organisms are called aposymbiotic organisms. The question is why some species can and others cannot establish the endosymbiotic relationship. The capability of algae for symbiosis with green hydra G. Kovacˇevic´ (&) S. Radic´ B. Jelencˇic´ M. Kalafatic´ correlates with the tolerance to low pH. A few hours after B. Pevalek-Kozlina the infection, pH of perialgal vacuoles is 3.5–4 (Huss et al. Division of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia 1993/1994). Symbiotic algae are acid-tolerant. Below pH e-mail: [email protected] 4, green algae constantly produce an increased amount of maltose (Kessler et al. 1991). Algae are mostly placed into H. Posilovic´ a quiet part of the cell where no intensive intracellular Department of Geology, Faculty of Science, University of Zagreb, Horvatovac 102a, decomposition occurs (McAuley and Smith 1982). 10000 Zagreb, Croatia Nonviable algae merge with the digestive vacuoles of 123 34 Author's personal copy G. Kovacˇevic´ et al. gastrodermal myoepithelial cells (O’Brien 1982). Endo- substrates. Generally, these enzymes have a broad spec- symbiosis with one algal species established once pre- trum of substrates and act on a variety of natural and cludes the same process with another species (Rahat 1991). xenobiotic compounds (Cummins et al. 2001). Naphthy- Green hydra is the only hydra host of zoochlorellae. lacetates (1- and 2-) used for esterase activity and visual- It has been considered that endosymbiotic algae from ization in tested Chlorella species are substrates of green hydra belong to the Chlorella genus, a huge and arylesterases (EC 3.1.1.2) and of carboxylesterases (EC complex genus of polyphyletic origin (Huss et al. 1993/ 3.1.1.1) (De Carvalho et al. 2003). The esterase zymograms 1994). Algae of the Chorella genus are morphologically contain a characteristic pattern of a large number of bands indistinguishable (Habetha et al. 2003). Particular species controlled by several loci (Cubadda and Quattrucci 1974). also belong to the class Chlorophyceae (Friedl 1997). They have been used often to study intra- and interspecific About 350 genera and 10,000 species of green algae have polymorphism (Brown and Weir 1983). been described (Ma¨dgefrau and Ehrendorfer 1988). Species Thus, the objective of the present study was to charac- closely related to Chlorella vulgaris Beij. (K&H, 1992), terize the endosymbiotic algae isolated from green hydra such as Chlorella kessleri Fott et Novak. (K&H, 1992), based on morphological, cytological, and isoenzyme anal- belong to the class Trebouxiophyceae and only those rep- ysis and to compare them with the two reference free-living resent the true genus Chlorella. Other species within Chlorella species. Chlorella not so closely related to the mentioned species or those included into other developmental lines within Trebouxiophyceae and Chlorophyceae should be included Materials and methods in another genus (Friedl 1997). Cytological morphometric analysis has been widely In the experiments, the following test organisms were used: used as a scientific method in biology and biomedicine Chlorella kessleri Fott et Novak. (K&H, 1992) strain (Agostinucci et al. 2002; Baron et al. 2004; Tonar and LARG/1 and Chlorella vulgaris Beij. (K&H, 1992) strain Markos 2004), in taxonomy (Nakahara et al. 2003), and SAG 211-11b, and endosymbiotic algae isolated from recently in symbiotic relationship research (Handa et al. green hydra. 2006; Kaltenpoth et al. 2005; Kovacˇevic´ et al. 2005). Algae were maintained on the surface of a sterile deep One of the advantages of using isoenzymes to study stock agar according to Pratt (1941) and modified accord- polymorphism is that a range of enzyme loci on one indi- ing to Horvatic´ et al. (2000). vidual can be studied easily using a small quantity of material with minimum preparation and cost. Morphological and cytological characterization Peroxidases (POX, EC 1.11.1.7) are ubiquitous enzymes found in virtually all green plants, many fungi, and aerobic Morphological and cytological characterization of endo- bacteria. The isozymic heterogeneity of peroxidases symbiotic algae isolated from green hydra in comparison appears to result from de novo synthesis, as well as an array with aposymbiotic standard species Chlorella vulgaris and of physiological/ecological determinants including hor- Chlorella kessleri was performed with binocular light mones, light, gravity, and infection (Siegel 1993). Peroxi- microscopes (Reichert and Nikon Eclipse E600, pro- dases have phylogenetically correlated similarities based gramme Lucia G DXM1200 version 4.81, camera Nikon on the chemical nature and redox potentials of the sub- DXM1200). For further morphological analysis, fresh strates they can oxidize. They catalyze the dehydrogena- algae were fixed onto the holder and coated with carbon tion of structurally diverse phenolic and endiolic substrates using sputter coater Bal-Tec SCD 050. The SEM micro- by H2O2 and are thus often regarded as antioxidant graphs were recorded using SEM Tescan TS 5136 MM in enzymes, protecting cells from the destructive influence of secondary electron mode at an accelerating voltage of

H2O2 and derived oxygen species (Vianello et al. 1997). 30 kV. The measured and compared parameters were cell In many plants, the tetrameric heme-containing enzyme area, cell radius, and chloroplast area. Measurements were catalase (CAT, EC 1.11.1.6) forms cellular populations performed on a sample of 200 cells for cell area and radius consisting of multiple forms (isoforms), which are usually and 50 cells for chloroplast area. The experiment was located in peroxisomes. Catalase, which degrades H2O2 performed in triplicate. into water and oxygen, is one of the major antioxidant enzymes. Three CAT isoforms have been identified in Enzyme extraction and soluble protein determination maize on separate chromosomes; they are differentially expressed and independently regulated (Scandalios 1990). Prior to homogenization, samples (150–330 mg) of Chlo- Esterases (EST), a group of hydrolases, catalyze the rella kessleri (Ck), Chlorella vulgaris (Cv), and endosym- formation or cleavage of ester bonds of water-soluble biotic algae isolated from green hydra (Cx) were scraped off 123 Morphological features and isoenzyme characterization Author's of endosymbiotic personal alga copy 35 the agar surface, collected in tubes, frozen in liquid nitrogen, Activity gel analysis and homogenized (TissueLyser, Qiagen) for 1 min at 30,000 Hz. Additional homogenization (1000) was performed Tissue extracts were run with anodic gel electrophoresis following addition of 500 ll of ice cold 50 mM potassium under nondenaturating conditions using vertical 10% (EST, phosphate (KPO4) buffer, pH 7.0, containing 1 mM EDTA POD) or 7% (CAT) polyacrylamide slab gels with the and 5 mM sodium ascorbate. The homogenates were then buffer system of Laemmli (1970). A constant voltage of centrifuged at 3,000g for 10 min at 4°C (Sigma 3K18 cen- 200 V was applied for 4 h and the temperature maintained trifuge). Supernatants were used for enzyme activity and at 4°C. Equal amounts of protein were loaded onto each isoenzyme and soluble protein analysis. When determining lane. For detection of POX isoforms, the gels were first the total amount of algae needed for isolation of soluble soaked in 50 mM sodium phosphate buffer (pH 7.0) for proteins, it was necessary to take a much smaller amount of 30 min and the bands visualized in the same buffer con- endosymbiotic algae isolated from green hydra, almost one- taining 4 mM H2O2 and 20 mM pyrogallol (Mittler and third to one-half less than the other samples. Soluble protein Zilinskas 1993). content was determined by the method of Bradford (1976) Staining for CAT isoenzymes was carried out as using bovine serum albumin (BSA, Sigma) as standard. described by Woodbury et al. (1971). Gels were washed in distilled water (3 9 15 min) and soaked in 5 mM H2O2 Enzyme assays (10 min). After a brief rinse, the gels were incubated in 1% (w/v) ferric chloride/1% potassium ferricyanide solution The activity of nonspecific peroxidase was measured by and CAT isozymes appeared as colorless bands on a dark monitoring the formation of purpurogallin at 430 nm green field. (e = 2.47 mM-1 cm-1) according to Chance and Maehly The esterase isozymes were visualized according to a

(1955). The reaction mixture contained 50 mM KPO4 modified procedure described by Balen et al. (2003). buffer (pH 7), 1 mM H2O2, 20 mM pyrogallol, and enzyme 1-Naphthyl acetate and 2-naphthyl acetate were dissolved extract. Catalase activity was determined by the decom- in 50% (v/v) acetone and mixed with 50 mM Tris/HCl, pH position of H2O2 and was measured spectrophotometrically 7.1. After staining (30 min), the gels were washed with tap by following the decrease in absorbance at 240 nm (Aebi water and incubated in a solution containing 50 mM 1984). Activity was calculated using the extinction coef- Tris/HCl, pH 7.1, and Fast Blue RR salt until dark brown ficient (e = 40 mM-1 cm-1) and a unit of CAT was (1-naphthyl acetate) or purple red (2-naphthyl acetate) defined as micromoles of decomposed H2O2 per gram of bands appeared (20–30 min). The Fast Blue RR salt was dry weight (DW) per minute. Esterase activity was deter- dissolved in absolute methanol and filtered into 50 mM mined spectrophotometrically at room temperature (25°C) Tris/HCl, pH 7.1. The gels were rinsed once more with tap using either 1- or 2-naphthylacetate as substrate (Burlina water and fixed in 30% (v/v) ethanol. and Galzigna 1972). Esterase activity was calculated fol- lowing the increase in absorbance at 322 nm (for 1-naph- thylacetate) or 313 nm (for 2-naphthylacetate), due to the Results -1 -1 formation of 1-naphtol (e322nm = 2.0 mM cm )or -1 -1 2-naphtol (e313nm = 1.25 mM cm ) (Balen et al. 2003). Qualitative characterization The reaction mixture contained 100 mM Tris/HCl, pH 7.4, and 100 mM 1- or 2- naphthylacetate, both dissolved in Endosymbiotic algae isolated from green hydra were of absolute methanol. The esterase activities were corrected coccoidal shape from the 1st to the 40th generation and of for spontaneous hydrolysis of 1- and 2-naphthylacetate. light green color. Cells divided into up to four autospores. The specific enzyme activity for all enzymes was From the 41st generation, algae started to grow in colo- expressed as units per milligram of protein (1 U = lmol nies and did not separate after division. Algae formed min-1). coenobia of up to four cells. Some of those showed empty cell lumens and were less viable or died. From the 43rd Statistical analysis generation, algae completely changed their morphology from coccoidal to elliptical coenobial shape (Fig. 1). Cells For each analysis, data were compared by analysis of in transition were also present. From the 46th generation, variance (ANOVA), using STATISTICA 8.0 (StatSoft, algae formed coenobia of up to seven cells. SEM con- USA) software package, and differences between corre- firmed the elliptical shape of the cells forming coenobia sponding controls and exposure treatment were considered (Fig. 2). as statistically significant at p \ 0.05. Each data point is In addition to their different shades of green color, the average of three replicates unless stated otherwise. endosymbiotic algae isolated from green hydra appeared to 123 36 Author's personal copy G. Kovacˇevic´ et al.

Fig. 1 Endosymbiotic alga isolated from green hydra. a 10–15 days after isolation. b 41st generation, the beginning of coenobia growth (arrows). c 43rd generation, coenobial growth (arrow) and changed algal morphology from coccoidal to elliptical form (2 arrows). Bar 5 lm

be noticeably more dry and of more granular structure than Morphometric characterization the other samples, enabling their easier handling and identification. Three morphometric parameters of tested Chlorella speci- mens were measured: cell area, cell radius, and chloroplast area (Table 1). Chlorella kessleri (Ck) and endosymbiotic algae isolated from green hydra (Cx) showed similar values for cell and chloroplast areas, whereas 50% smaller values of the parameters were found for Chlorella vulgaris (Cv). Regarding cell radius, values declined in the order Ck [ Cx [ Cv.

Peroxidase activity and isoenzyme analysis

The highest POX activity was observed in the Ck sample (Fig. 3a). Cx and Cv samples exhibited 34 and 63% lower activity, respectively, in comparison with the Ck sample. Native electrophoresis revealed five isoperoxidases in tested Chlorella samples (Fig. 3b). Four isoenzymes (POX1-4) were noted in the Ck sample, three (POX2, 4,

Table 1 Morphometric analysis of Chlorella vulgaris (Cv), Chlo- rella kessleri (Ck), and Chlorella isolated from green hydra (Cx) Cell area Cell radius (lm) Chloroplast area

Cv 6.31 ± 0.12 1.28 ± 0.014 2.98 ± 0.059 Ck 12 ± 0.27 2.24 ± 0.017 8.22 ± 0.144 Fig. 2 SEM of endosymbiotic alga isolated from green hydra (43rd Cx 12.95 ± 0.26 2.17 ± 0.030 8.83 ± 0.160 generation). Bar 5 lm

123 Morphological features and isoenzyme characterization Author's of endosymbiotic personal alga copy 37

(A) (B) POX1 EST1 0.8 POX2 (A) (B) EST 2 POX3 ) POX4 EST 3 -1 EST 4 0.6 EST 5 EST 6 POX5 EST 7 2.5 EST 8 0.4 EST 9 1-naphtyl acetate 2-naphtyl acetate EST 10 )

-1 2.0 EST 11 EST 12 0.2 EST 13 EST 14

POX (U mg protein 1.5 EST 15 EST 16 0 EST 17 1.0 EST 18 Cv Ck Cx Cv Ck Cx 0.5 EST 19

EST (U mg protein EST 20 Fig. 3 Peroxidase (POD) activity (a) and isoenzyme analysis (b)in EST 21 Chlorella vulgaris (Cv), Chlorella kessleri (Ck), and endosymbiotic 0.0 Cv Ck Cx Chlorella isolated from green hydra (Cx). Values are mean ± SD Cv Ck Cx based on three replicates Fig. 5 Esterase (EST) activity (a) and isoenzyme analysis (b)in and 5) in the Cv sample, while in the Cx sample only Chlorella vulgaris (Cv), Chlorella kessleri (Ck), and the endosym- POX2 was expressed. biotic Chlorella isolated from green hydra (Cx). Values are mean ± SD based on three replicates

Catalase activity and isoenzyme analysis Chlorella species assayed with 2-naphthylacetate signifi- cantly declined in the order Ck [ Cx [ Cv. As in the case of POX, CAT activity was highest in the Ck Results presented in Fig. 5b show the variations in the sample (Fig. 4a). Cv and Cx samples showed 56 and 79% esterase isoenyzme pattern of Chlorella vulgaris (Cv), lower activity, respectively, in comparison with the Ck Chlorella kessleri (Ck), and the endosymbiotic Chlorella sample. Four anodic CAT isoenzymes were observed in isolated from green hydra (Cx). The esterases were num- tested Chlorella samples (Fig. 4b), of which all four were bered in sequence starting from the anode, according to present in the Ck sample, three (CAT1, 3, and 4) in the Cv their decrease in negative charge. In total, 21 esterase sample, and only one (CAT1) in the Cx sample. isoenzymes were resolved in three algal samples but only isoesterases EST4 and 5 were common to all samples. In Esterase activity and isoenzyme analysis addition to those, isoesterases EST10-12 were expressed in the Cv sample (five in total). Seven additional isoenzymes, Esterase activity was evaluated with two substrates, 1- and EST8 and EST13-18, were expressed in the Ck sample 2-naphthyl acetate. The enzyme activity of the Ck sample only. Electrophoretic pattern of the Cx sample revealed 14 measured with 1-naphthylacetate was considerably higher isoesterases in total: 9 isoesterases (EST1-3, EST6-7, than in the Cv sample, but the highest activity was found in EST9, EST19-21) not present in either of the referent the Cx sample (Fig. 5a). The esterase activity of tested Chlorella (Cv and Ck) samples, 3 isoesterases present in the Cv sample (EST10-12) but not in the Ck sample, and 2 isoesterases common to all tested algae (EST4-5). The differential staining pattern showed that EST7, (A) (B) CAT1 EST 9, EST11-12, EST14, and EST19 isoenzymes hydro- 0.12 CAT2 lyze 1-naphthyl acetate; EST1-4, EST6, EST13, EST16, ) CAT3 -1 EST18, and EST20 isoenzymes hydrolyze 1-naphthyl 0.08 CAT4 acetate, and EST5, EST8, EST10, EST15, EST17, and EST21 isozymes hydrolyze both 1- and 2-naphthyl acetate.

0.04

CAT (U mg protein Discussion 0 Cv Ck Cx Cv Ck Cx Morphometric characteristics are quantitative characteris- tics that have a complex genetic basis, with the environ- Fig. 4 Catalase (CAT) activity (a) and isoenzyme analysis (b)in ment exerting a strong influence on phenotypic variations Chlorella vulgaris (Cv), Chlorella kessleri (Ck), and the endosym- biotic Chlorella isolated from green hydra (Cx). Values are within populations (Falconer 1986; Lewontin 1974). mean ± SD based on three replicates According to Fawley et al. (2004), unicellular green algae 123 38 Author's personal copy G. Kovacˇevic´ et al. display characteristics of environmental plasticity. If sur- Endosymbiotic alga displayed specific green coloring and vival capacity is increased by a changed phenotype, mor- appeared drier and more granular than the other samples and, phological plasticity can provide a selective advantage as such, could easily be recognized among the samples. In (Diaz-Pulido et al. 2007). addition, compared to the reference Chlorella species, much In the case of green hydra symbiosis, only unicellular smaller quantities (almost one-third to one-half) of endo- symbiosome formation is possible inside the gastrodermal symbiotic algae were required for the soluble protein anal- myoepithelial cells. After their isolation, endosymbiotic ysis. A lateral eukaryote/eukaryote gene transfer has algae became aposymbiotic, and their plasticity changes recently been suggested between hydra and endosymbiotic from coccoidal to elliptical form. Almost 3 years after the algae (Habetha and Bosch 2005). It has also been noticed culture initiation, all isolated aposymbiotic algae displayed that some algae are placed very close to the host nucleus the elliptical shape. Isolated algae that could not follow the (Habetha et al. 2003). This position might have enabled plasticity transformation into a coenobial form lost almost mutual transfer of genetic material between hydra and 100% of their viability. Hegewald and Hanagata (2000) endosymbiotic algae. A clearer insight into the phylogenetic showed that the coenobial ellipsoid Dicloster acuatus, status of hydras themselves, maintenance of symbiosis and formerly classified within the family Scenedesmaceae, is course of symbiogenesis in green hydra is desirable. more related to Chlorella kessleri. In conclusion, symbiosis can significantly alter life on Phenotypic plasticity is estimated by the number of cells Earth. Symbiosis is not a side or a sporadic event. Sym- in one coenobium. It is not clear which mechanisms biotic organisms are abundant, diverse, and significant. determine the number of cells per coenobium. Our isolated This significance is characterized by biodiversity, regula- symbionts consisted of up to seven cells per coenobium, tion, maintenance, and improvements of the symbiotic which probably represents the plasticity pattern needed for mechanisms in the present association. Symbiosis is survival of the aposymbiotic system. Based on morpho- an important biological principle whose recognition is metric parameters, higher similarity was noticed between unavoidable for understanding of the living world. Con- the isolated endosymbionts and C. kessleri than between cerning the morphometric similarity of isolated algal the isolated endosymbionts and C. vulgaris. endosymbionts with the free-living relative C. kessleri and In general, changes in the isoenzyme (CAT, POX, EST) isozyme similarity with the free-living relative C. vulgaris, patterns were consistent with the quantitatively measured it could be concluded that symbiogenesis in green hydras is enzyme activities in tested algae. Results obtained by the an ongoing process. native anodic electrophoresis suggest complex relations within the Chlorella genus. The results of CAT and POX Acknowledgments The presented results are a product of the sci- isoenzyme analysis indicate that the endosymbiotic Chlo- entific project ‘‘Molecular phylogeny, evolution and symbiosis of freshwater invertebrates’’ and project 119-1191196-1202 carried out rella isolated from green hydra is equally related to both with the support of the Ministry of Science, Education and Sport of reference algae from Chlorella genus. However, based on the Republic of Croatia. the huge esterase polymorphism, a higher similarity was observed between the isolated endosymbiotic Chlorella and Chlorella vulgaris than between the former and References Chlorella kessleri. In addition, nine EST isoenzymes were specifically expressed in the isolated endosymbiotic alga, Aebi H (1984) Catalase in vitro. 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Baron PJ, Real LE, Ciocco NF, Re ME (2004) Morphometry, growth and reproduction of an Atlantic population of the razor clam Due to the high polymorphism of EST isoenzymes in the Ensis macha. Sci Mar 68:211–217 tested species, esterases could serve as useful genetic Beach MJ, Pascoe D (1998) The role of Hydra vulgaris (Pallas) in indicators for identification of different Chlorella species. assessing the toxicity of freshwater pollutants. Wat Res 32:101– With long-term maintenance of cultures, it became 106 Bradford MM (1976) A rapid and sensitive method for the obvious that endosymbiotic alga from green hydra were quantitation of microgram quantities of protein utilizing the morphologically different from the reference species. principle of protein-dye binding. Anal Biochem 72:248–254 123 Morphological features and isoenzyme characterization Author's of endosymbiotic personal alga copy 39

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