Microb. Ecol. 7:183-193 (1981)
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Effects o f C a d m i u m a n d C o p p e r on the Ultrastructure o f A n k i s t r o d e s m u s braunii a n d A n a b a e n a 7120 A. Massalski*, V. M. Laube**, and D. J. Kushner Department of Biology, University of Ottawa, Ottawa, Ontario K 1N 6N5, Canada
Abstract. The effects of brief exposure to, or growth in the presence of, lethal and sublethal concentrations of Cu(NO)2 and Cd(NO3) on the ultrastructure of the blue-green alga Anabaena 7120 and the green alga Ankistrodesmus braunii were studied. Exposure to increasing amount of both metal ions led to the appearance of larger proportions of electron-dense cells whose organelles were less well defined than those of untreated cells. Metal-treated cells of Anabaena 7120 became distorted. Some had a corrugated appearance. Others lysed, leaving a much larger proportion of heterocysts. Such heterocysts were often empty or had a curious collapsed appearance. Growth ofA. braunii in the presence of 10 -4 M Cu(NO3)2 produced substantial numbers of multinucleate giant cells with thick walls; such cells result from repeated mitotic division without subsequent cytokinesis. The giant ceils contained centrioles, structures not as yet found in normal cells of the genus Ankistrodesmus. Some nuclei of giant, but not of normal, cells contained deep indentations that appeared as "holes" in cross section. Some giant cells also contained triple parallel strands of endoplasmic reticulum which extended across much of the cell, connecting to the nuclear envelope. Some ultrastructural changes were also noted in algal cells grown over sediment containing Cu or Cd, but these were generally less severe than those occurring when metal ions were added directly to the algal cultures.
Introduction Past studies of the effects of toxic metals on algae have been concerned mainly with growth rate [I, 5, 19], detoxification processes [2], and photosynthesis [10, 20]. Only a few studies of the effects of toxic metals on algal morphology have been carded out. Nuzzi [8], using light microscopy, showed that the marine diatom Phaeodactylum tricornutum became highly vacuolated and assumed an ovoid shape when grown in the presence of mercury. Under similar conditions, a Chlorella sp. formed giant, morphologically aberrant cells. Silverberg [16] found intramitochondrial granules in cells of Ankistrodesmusfalcatus exposed to Cd 2+ and conf'trmed, using energy-dispersive *Present address: Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada N6A 5C1. **Present address: Division of Biological Science, National Research Council, Ottawa, Ontario, Canada K1A 0R6. Send offprint requests to D. J. Kushner at the above address.
0095-3628/81/0007-0183 $02.20 9 1981 Springer-Verlag New York Inc.
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analysis, that these granules contained Cd. Pb ( N O 3 ) 2 caused inflated and disorganized thylakoids in the chloroplasts of the green alga Stigeoctonium tenue [15]. Hg CI 2 and phenyl mercuric acetate caused numerous ultrastructural changes in Anabaena flosaquae and Scenedesmus sp. [ 12]. Studies of the toxicity and binding of Cu 2§ Cd 2+, and Pb 2+ ions to the blue-green alga Anabaena 7120 and the green alga Ankistrodesmus braunii have been reported [7]. The ultrastructural changes that occur when these algae are treated with Cu 2+ and Cd 2+ are described here.
Materials and Methods Detailed descriptions of algae, culture media, and growth conditions are given in [7], which also describes determinations of Cu and Cd bound to algae and sediments.
Cultures Anabaena 7120, initially obtained from Dr. R. Y, Stanier, Institut Pasteur, Pads, was maintained in BG-II medium (Stanier et al, [ 18]. Experiments with the alga, which can fix atmospheric nitrogen, were carried out in the same medium minus NaNO 3. Ankistrodesmus braunff (Naegeli) Collins was isolated from the Ottawa River, Canada. This species was maintained and studied in media previously described by Ohad et al. 19]. All cultures were grown at 20~ l~ under white fluorescent tubes (Westinghouse). Light intensity was maintained at 1300 lux with a 16 h-18 h light-dark cycle. Growth was measured in a Coleman Junior spectrophotometer as optical density (O.D.) at 650 nm, in a tube, 18 mm outside diameter.
Treatment of Algae with Toxic Metals The effects of Cu 2+ and Cd 2+ ions on algal growth have been described in detail [7]. This work showed that the presence of sublethal concentrations of such metals increased the lag phase of cultures ofAnabaena 7120. In the present experiments Cu 2+ or Cd 2+ ions, at the concentrations indicated, were added to growing cultures of an O,D.650 am of 0,50. After I hour cells were harvested and fixed for electron microscopic examination, Cu 2 + and Cd 2 + decreased the growth yield ofA. braunff without increasing the lag phase [7]. In the present experiments A. braunii was grown in the indicated metal ion concentrations and harvested late in growth: O,D. 650 nm = 1.90 in the untreated culture, 0.70 in I 0 - 5 M Cu(No 3) 2.0.50 in 10 - 4 M Cu(NO 3) 2, and 0.42 in 10 - 5 M Cd(NO3)2; no growth took place in 10 - 4 M Cd(NO3) 2. Experiments in which algae were grown in the presence of Cu- or Cd-containing sediment have been previously described [7], All cultures ofA. braunii were harvested at O.D. 650 nrn of 1.90-2. I0. Cultures of Anabaena 7120 were harvested at O.D. 650 nm values of 1.90 for the control (cultures grown in the absence of sediment), and 0.78 and 0.82 for cultures grown in the presence of Cd- and Cu-loaded sediments, respectively, As previously shown [6, 7] the unloaded sediment already contained 50 ppm Cu and no detectable Cd.
Electron Microscopy Cells were harvested by centrifuging in a clinical centrifuge and then fixed at room temperature for 1 h in 3% glutaraldehyde in 0.05M phosphate buffer at pH 7. I. The cells were then washed three times in buffer, postfixed for I h in cold 1% OsO 4, and then dehydrated in a graded acetone series. The dehydrated cells were
Effects of Metal Ions on Algal Ultrastructure
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Table I. Distribution of different types of cells in cultures of Anabaena7120 treated with Cu and Cd
Treatment Untreated culture 10 -5 M Cu(NO3) 2 l0 -4 M Cu(NO3) 2 10-5 M Cd(NO3) 2 10-4 M Cd(NO3)2 Grown over Cu-loaded sediment Grown over Cd-loaded sediment
Cu or Cd Vegetative cells a bound (/ag/mg cells) b Normal Distorted
Heterocysts a Normal
Distorted
-7 N.D. 70 N.D. 0.004
92.5 89.0 1.6 3.2 0 90.4
0 2.6 20.9 84.9 82.7 0.7
7.5 4.2 5.4 0 1.8 6.7
0 4.2 72.0 11.9 15.5 2.2
0.058
27.3
61.2
1.4
10.1
a Figures show percent of each kind of cell. From 114 to 263 (usually ca. 130) cells were examined after each treatment. More details on the kinds of distortions observed are given in the text. bEstimated from the results of Laube et al. [7] or determined separately for these experiments as described in [7]: substantially higher amounts bound would be expected with the higher Cu or Cd concentrations, although these were not determined (N. D.).
infiltrated with Spurr's hard resin mixture [17], which was then polymerized at 60~ for 18 h. Silver- and gold-colored sections were cut with a DuPont diamond knife on a Sorval Porter-Blum MT-2B ultramicrotome, mounted on acetone-treated 200 mesh copper grids without support, and stained for 5 rain in uranyl acetate (5% in 50% ethanol) followed by 3 min in lead citrate [ 13]. Observations and photographs were made with a Philips 201 electron microscope.
Results and Discussion
Morphological Changes of Anabaena 7120 V e g e t a t i v e cells (Fig. 1) and heterocysts (Fig. 2) of Anabaena 7120 f r o m untreated cultures s h o w m o r p h o l o g i c a l features typical of this genus o f b l u e - g r e e n algae [14]. U n t r e a t e d cultures consisted entirely o f normal cells plus about 8% normal heterocysts (Table 1). C u l t u r e s treated with 10 -5 M C u ( N O 3 ) 2 s h o w e d little m o r p h o l o g i c a l change. W h e n the culture was treated with 1 0 - 4 M C u ( N O 3 ) 2, considerable lysis occurred, as s h o w n by the d e c r e a s e in turbidity and appearance o f blue p h y c o c y a n i n pigment in solution. This is r e f l e c t e d in the m u c h smaller proportion o f cells to heterocysts (Table 1). This must have resulted f r o m selective b r e a k d o w n o f vegetative cells with better preservation o f the t h i c k e r - w a l l e d heterocysts. H o w e v e r , m o s t heterocysts w e r e distorted, as were the r e m a i n i n g ceils. Cells with a corrugated appearance, mostly darkly stained (electron dense), w e r e the m o s t c o m m o n f o r m , although s o m e lighter corrugated cells of normal e l e c t r o n density w e r e also o b s e r v e d (Figs. 3 and 4). M o s t distorted heterocysts had a c o l l a p s e d appearance, and m a n y were empty. T h e degree to w h i c h heterocysts were c o l l a p s e d varied: s o m e w e r e indented or partly collapsed. Others were almost
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Fig. 1. Vegetative cell ofAnabaena 7120, untreated culture. Cg, cyanophycin granules; PB, polyhedral body; thylakoids (arrow). 22,500X. Fig. 2. Anabaena 7120 heterocyst, untreated culture. 18,000X.
Fig. 3. Anabaena 7120 vegetative cell grown over Cd-loaded sediment. Note the corrugated appearance of the cell. Is, intrathylakoidal space. 27,000X. Similar cells were seen after Cu 2+ addition to cultures. Fig, 4. Anabaena 7120 vegetative cell grown over Cd-loaded sediment. Note the much darker appearance of the cell compared with Fig. I. Is, intrathylakoidal space. 28,500X. Similar cells were seen after Cu 2+ or Cd 2+ addition to cultures.
Effects of Metal Ions on Algal Ultrastructure
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Fig, 5. Anabaena 7120 heterocyst. This cell had collapsed in such a way that in a cross sectionit appears hollow, Culture treated with 10-4M Cu 2+. 25,000X, Fig. 6, Anabaena 7120 heterocyst, showing partiallydisintegratedcell wall, Culturetreated with 10 -4M Cu 2+ . 21,000X. Fig. 7. Anabaena 7120 heterocyst, showingcompletelydisintegratedcell wall. Culturetreated with 10-4M Cu 2+, 21,400. Fig. 8. Anabaena 7120. A fieldof deformed cells. Culturetreated with 10 -SM Cd2+ . 4,200X.
completely flattened so that the cell contents were practically indistinguishable. That shown in Fig. 5 is so deeply indented that it appears hollow in cross section. In a number of half-empty heterocysts the cell walls had become networks of fine, electron-dense fibrils. That shown in Fig. 6 is partly fibriUar, whereas that in Fig. 7 is completely enclosed by a thick, diffuse layer of electron-dense fibers. It is uncertain if
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these fibers arise from the disintegrating cell wall or if they are synthesized as the wall breaks down. In contrast to Cu (NO3)2, Cd(NO3) 2 caused no visually observable lysis. The ratio of cells to heterocysts decreased only slightly (Table 1). Treatment with 10 -5 o r 10 -4 M Cd(NO3)2 distorted most or all the cells, leading mainly to the kind of highly electrondense, corrugated appearance shown in Fig. 4. An unusual type of shape distortion, found only in cells treated with 10 -5 M Cd(NO3) 2, is shown in Fig. 8. These cells were partly flattened in such a way that in sections at least one part of the cell appeared to have a straight edge. Most of the heterocysts were distorted; half-empty ones, as shown in Fig. 6, predominated. In cultures grown over Cu-loaded sediment, no significant change in the proportion of normal cells occurred. Some cells, however, had numerous intrathylakoidal spaces (Fig. 9), a feature rarely encountered in untreated cells. Centrally located empty spaces, which often contained polyhedral bodies, were observed in this experiment but never in untreated cultures. Polyhedral bodies were also more numerous than in untreated cells. Most heterocysts appeared normal, with only a small proportion of empty ones. In contrast, much more cell distortion was seen in cultures grown over Cd-loaded sediment (Table 1; Figs. 3 and 4), with corrugated cells, both highly electron dense and of normal electron density, predominating and roughly equal numbers of collapsed and empty heterocysts.
Morphological Changes in Ankistrodesmus braunii Figure 10 shows, in cross section, a young cell ofA. braunii from an untreated culture. The cell is still enclosed by the mother cell wail. Organelles characteristic of a chlorococcalean alga are seen: a chloroplast with a single pyrenoid; a nucleus with adjacent Golgi apparatus; and mitochondria, endoplasmic reticulum (ER), and ribosomes. In such untreated cultures, harvested in late exponential growth, about one-third of the cells appeared similar to that shown in Fig. 10, with all organelles clearly discernible. More than half the cells were more electron dense, but the large cell organelles---particularly the pyrenoid----could still be distinguished. The proportions of cell types remained roughly the same in cells grown in the presence of 10-5 Cu(NO3)2 (Table 2). When cells were grown in the presence of 10-4M Cu(NO3) 2, many were so electron dense that not even the pyrenoid was recognizable. The most striking result of treatment with high Cu 2+ concentration was the presence of multinucleate giant cells (Figs. 11-14). These cells presumably resulted from repeated mitotic division without subsequent cytokinesis. Some, however, were undergoing cytokinesis (Fig. 11). We could not determine what fraction of such cells finally undergo cytokinesis or remain multinucleate. In fact, most giant cells were so electron dense that it was difficult to know what division processes had occurred. In addition to the multinucleate giant cells, other giant cells had several daughter cells, probably in multiples of four, still enclosed by a mother cell wall. Figure 15 shows an electron-dense cell containing at least 12 daughter cells. The walls of giant cells were considerably thicker than those of normal mother cells. An unusual feature of some giant cells, never seen in untreated cultures, consisted of three parallel strands of endoplasmic reticulum connected to the nuclear envelope and traversing at least half the cell length (Fig. 12). A section of a nucleus with a number of
Effects of Metal Ions on Algal Ultrastructure
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Fig. 9. Anabaena 7120 vegetative cell grown over Cu-loaded sediment. PB, polyhedral body; Is intrathylakoidal space. 32,000X. Fig. 10. Ankistrodesmus braunii, untreated culture showing the main cell organelles. N, nucleus; G, Golgi apparatus; P, pyrenoid;m, mitochondrion.30,500X. deep indentations is shown in Fig. 13. Such indentations were also found only in giant cells, and it is uncertain if such nuclei can subsequently divide. Pickett-Heaps [11] suggests that in the genus Ankistrodesmus centrioles are transitory structures, appearing only during mitosis and cell cleavage. We have found centrioles in the giant cells of A. braunii. Two of them, partly enclosed by the nucleus, are seen in Fig. 14.
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Fig. 11, Ankistrodesmus braunii, A giant cell showing 5 nuclei (N). The cell is undergoing cytokinesis as indicated by cleavage furrows (arrows). Culture treated with 10 -4M Cu 2 + 10,000X, Fig. 12. Ankistrodesmus braunii. Giant cells, showing part of the nucleus (N) and 3 parallel strands of endoplasmic reticulum (ER) connected with the nuclear envelope (arrows). Culture treated with 10-4M Cu 2+. 27,000X. Fig. 13. Ankistrodesmus braunii. A large nucleus (N) of a giant cell showing 2 deep indentations which in a cross section appear hollow. Culture treated with 10 -4M Cu 2+ . 26,000X. Fig. 14. Ankistrodesmus braunii giant cell showing 2 centrioles (arrows) adjacent to the nucleus (N). Note the thick cell wall (CW). Culture treated with t0 -4M Cu 2+. 49,500X.
Effects of Metal Ions on Algal Ultrastructure
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Table 2. Distributionof differenttypes of cells in culturesofA. brauniitreated withCu and Cd
Treatment Untreatedculture 10 -5 M Cu(NO3)2 10 -4 M Cu(NO3)2 10-5 M Cd(NO3)2 Grown over unloded sedimentc Grown over Cu-loaded sediment Grown over Cd-loaded sediment
Cu or Cd bound (tag/mg cells)b
Normal cells a
Very electron dense cellsa
Giant cellsa
Dead ceUsa
-0.7 3.0 1.4 0.016
90.2 95.4 3.5 0 95.7
0 0 61.5 96.5 0
0 0 26.5 0 0
9.8 4.6 8.4 3.5 4.3
0.04
98.2
0
0
1.8
0.025
93.7
0
0
6.3
aFigures show percentof each kind of cell. From 142 to 329 (usuallyca. 220) cells were examinedafter each treatment. Both more- and less-electron-densecells are includedin the countsof normaland giant cells (see text). Cells partlyor entirelyemptyof internalcontentsare considered"dead." Veryelectron-densecellswere so dark that no internalstructureswere visible. bFrom the resultsof Laubeet al. [7] or determinedseparatelyfor these experiments. cThis sedimentalreadycontained50 ppm Cu [6].
After growth in the presence of 10-5 M Cd(NO3)2, practically all (96.5%) of the cells were very electron dense, with virtually indistinguishable cell organelles (Fig. 16). No giant cells were found. As previously pointed out [7], the presence of Cu- or Cd-loaded sediment did not inhibit the growth o f A . braunii, even though some of the metals became bound to the algal cells. No significant change of morphology was seen after growth in the presence of sediment (Table 2); cells that were electron dense still had discernible organelles. Although the sites of Cu and Cd action on both species are not known, our results suggest that they may act on the outer cell layers. The lysis of vegetative cells of Anabaena 7120 by Cu 2+ may reflect an action on the cell walls. The corrugated appearance caused by Cu 2+ and Cd 2+ may be due to a direct action on the walls, o r t o an osmotic effect of the f'Lxative on cells with weakened walls. The unusual distortion of cells exposed to 10-5 M Cd (Fig. 8) could also be due to an effect on wall rigidity that causes cells to flatten against each other during centrifugation. The inhibition by Cu 2+ of cytokinesis and/or cell separation in A. braunii might also reflect an action o f this metal on the cell's outer surface. Cu 2+ can inhibit cell division in Chlorella pyrenoidosa [ 19] and C. ellipsoidea [4], and can cause giant cell formation in C. vulgaris [3]. Mercury can also cause giant cell formation in a Ch'lorella sp. and the green flagellate Dunaliella tertiolecta [2]. It might be profitable for further studies of such metals to concentrate on the cell surface as a site of action.
Acknowledgments. This work was supported by grants from environmentCanada and The Natural Sciences and EngineeringResearch Councilof Canada.
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Fig. 15. Ankistrodesmus braunii dark giant cell showing at least 12 daughter cells enclosed by a mother cell wall. Culture treated with 10 - 4 M Cu 2+. 1 I, 100X. Fig. 16. Ankistrodesmus braunii dark cell. Note high electron opacity of the cell and lack of morphological details. Culture treated with 10 - 5 M Cd 2+ . 20,000X.
References I. Bartlett, L., F. W. Rabe, and W. H. Funk: Effects of copper, zinc and cadmium on Selenastrum capricornutum. Water Res. 8, 179-185 (1974) 2. Davies, A. G.: An assessment of the basis of mercury tolerance in Dunaliella tertiolecta. J. Mar. Biol. Assoc. U.K. 56, 39-57 (1976) 3. Foster, P. L.: Copper exclusion as a mechanism of heavy metal tolerance in a green alga. Nature 269, 322-323 (1977) 4. Kanazawa, T., and K. Kanazawa: Specific inhibitory effect of copper on cellular division in Chlorella. Plant Cell Physiol. 10, 495-502 (1960) 5. Klass, E., D. W. Rowe, and E. J. Massaro: The effect of cadmium on population growth of the green alga Scenedesmus quadricauda. Bull. Environ. Contam. Toxicol. 12, 442-445 (1974) 6. Laube, V. M., S. Ramamoorthy, D. J. Kushner: Mobilization and accumulation of sediment-bound heavy metals by algae. Bull. Environ. Contam. Toxicol. 21,763-770 (1979) 7. Laube, V. M., C. McKenzie, and D. J. Kushner: Strategies of algal dealings with heavy metals. Can. J. Microbiol. 26, 1300-1311 (1980) 8. Nuzzi, R.: Toxicity of mercury to phytoplankton. Nature 237, 38--40 (1972) 9. Ohad, I., P. Siekevitz, and G. E. Palade: Biogenesis of chloroplast membranes. J. Cell Biol. 35, 521-552 (1967) 10. Overnell, J.: The effect of some heavy metal ions on photosynthesis in a freshwater alga. Pesticide Biochem. Physiol. 5, 19-26 (1975) 11. Pickett-Heaps, J. D.: Green Algae--Structure, Reproduction and Evolution in Selected Genera. Sinaur Associates, Publishers, Sunderland, MA (1975)
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12. Ramamoorthy, S., A. Massalski, and T. Bistricki: Energy dispersive X-ray microanalysis of mercury in environmental samples. Proceedings of the Symposium on Electron Microscopy and X-ray Applications to Environmental and Occupational Health Analyses, Vol. I, pp. 247-254. Ann Arbor Sciences Publishers, Ann Arbor (1978) 13. Reynolds, E. S.: The use of lead citrate at high pH as an electron-opaque stain in bacteria. J. Bacteriol. 70, 691-701 (1963) 14. Shively, J. M.: Inclusion bodies in prokaryotes. Annu. Rev. Microbiol. 28, 167-187 (1974) 15. Silverberg, B. A.: Ultrastructural localization of lead in Stigecloniam tenue (Chlorophyceae, Ulotrichales) as demonstrated by cytochemical and X-ray microanalysis. Phycologia 14, 265-274 (1975) 16. Silverberg, B. A.: Cadmium-induced ultrastructural changes in mitochondria of freshwater green algae. Phycologia 15, 155-159 (1976) 17. Spurr, A. R.: A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) 18. Stanier, R. Y., R. Kunisawa, M. Mandel, and G. Cohen-Bazire: Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 31, 171-205 ( 1971) 19. Steeman Nielsen, E., and L. Kamp-Nielsen: Influence of deleterious concentrations of copper on the growth of Chlorellapyrenoidosa. Physiol. Plant. 23, 828-840 (1970) 20. Steeman Nielsen, E., and S. Wium-Anderson: The influence of Cu on photosynthesis and growth in diatoms. Physiol. Plant. 24, 480--484 (1971)
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Effects o f C a d m i u m a n d C o p p e r on the Ultrastructure o f A n k i s t r o d e s m u s braunii a n d A n a b a e n a 7120 A. Massalski*, V. M. Laube**, and D. J. Kushner Department of Biology, University of Ottawa, Ottawa, Ontario K 1N 6N5, Canada
Abstract. The effects of brief exposure to, or growth in the presence of, lethal and sublethal concentrations of Cu(NO)2 and Cd(NO3) on the ultrastructure of the blue-green alga Anabaena 7120 and the green alga Ankistrodesmus braunii were studied. Exposure to increasing amount of both metal ions led to the appearance of larger proportions of electron-dense cells whose organelles were less well defined than those of untreated cells. Metal-treated cells of Anabaena 7120 became distorted. Some had a corrugated appearance. Others lysed, leaving a much larger proportion of heterocysts. Such heterocysts were often empty or had a curious collapsed appearance. Growth ofA. braunii in the presence of 10 -4 M Cu(NO3)2 produced substantial numbers of multinucleate giant cells with thick walls; such cells result from repeated mitotic division without subsequent cytokinesis. The giant ceils contained centrioles, structures not as yet found in normal cells of the genus Ankistrodesmus. Some nuclei of giant, but not of normal, cells contained deep indentations that appeared as "holes" in cross section. Some giant cells also contained triple parallel strands of endoplasmic reticulum which extended across much of the cell, connecting to the nuclear envelope. Some ultrastructural changes were also noted in algal cells grown over sediment containing Cu or Cd, but these were generally less severe than those occurring when metal ions were added directly to the algal cultures.
Introduction Past studies of the effects of toxic metals on algae have been concerned mainly with growth rate [I, 5, 19], detoxification processes [2], and photosynthesis [10, 20]. Only a few studies of the effects of toxic metals on algal morphology have been carded out. Nuzzi [8], using light microscopy, showed that the marine diatom Phaeodactylum tricornutum became highly vacuolated and assumed an ovoid shape when grown in the presence of mercury. Under similar conditions, a Chlorella sp. formed giant, morphologically aberrant cells. Silverberg [16] found intramitochondrial granules in cells of Ankistrodesmusfalcatus exposed to Cd 2+ and conf'trmed, using energy-dispersive *Present address: Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada N6A 5C1. **Present address: Division of Biological Science, National Research Council, Ottawa, Ontario, Canada K1A 0R6. Send offprint requests to D. J. Kushner at the above address.
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analysis, that these granules contained Cd. Pb ( N O 3 ) 2 caused inflated and disorganized thylakoids in the chloroplasts of the green alga Stigeoctonium tenue [15]. Hg CI 2 and phenyl mercuric acetate caused numerous ultrastructural changes in Anabaena flosaquae and Scenedesmus sp. [ 12]. Studies of the toxicity and binding of Cu 2§ Cd 2+, and Pb 2+ ions to the blue-green alga Anabaena 7120 and the green alga Ankistrodesmus braunii have been reported [7]. The ultrastructural changes that occur when these algae are treated with Cu 2+ and Cd 2+ are described here.
Materials and Methods Detailed descriptions of algae, culture media, and growth conditions are given in [7], which also describes determinations of Cu and Cd bound to algae and sediments.
Cultures Anabaena 7120, initially obtained from Dr. R. Y, Stanier, Institut Pasteur, Pads, was maintained in BG-II medium (Stanier et al, [ 18]. Experiments with the alga, which can fix atmospheric nitrogen, were carried out in the same medium minus NaNO 3. Ankistrodesmus braunff (Naegeli) Collins was isolated from the Ottawa River, Canada. This species was maintained and studied in media previously described by Ohad et al. 19]. All cultures were grown at 20~ l~ under white fluorescent tubes (Westinghouse). Light intensity was maintained at 1300 lux with a 16 h-18 h light-dark cycle. Growth was measured in a Coleman Junior spectrophotometer as optical density (O.D.) at 650 nm, in a tube, 18 mm outside diameter.
Treatment of Algae with Toxic Metals The effects of Cu 2+ and Cd 2+ ions on algal growth have been described in detail [7]. This work showed that the presence of sublethal concentrations of such metals increased the lag phase of cultures ofAnabaena 7120. In the present experiments Cu 2+ or Cd 2+ ions, at the concentrations indicated, were added to growing cultures of an O,D.650 am of 0,50. After I hour cells were harvested and fixed for electron microscopic examination, Cu 2 + and Cd 2 + decreased the growth yield ofA. braunff without increasing the lag phase [7]. In the present experiments A. braunii was grown in the indicated metal ion concentrations and harvested late in growth: O,D. 650 nm = 1.90 in the untreated culture, 0.70 in I 0 - 5 M Cu(No 3) 2.0.50 in 10 - 4 M Cu(NO 3) 2, and 0.42 in 10 - 5 M Cd(NO3)2; no growth took place in 10 - 4 M Cd(NO3) 2. Experiments in which algae were grown in the presence of Cu- or Cd-containing sediment have been previously described [7], All cultures ofA. braunii were harvested at O.D. 650 nrn of 1.90-2. I0. Cultures of Anabaena 7120 were harvested at O.D. 650 nm values of 1.90 for the control (cultures grown in the absence of sediment), and 0.78 and 0.82 for cultures grown in the presence of Cd- and Cu-loaded sediments, respectively, As previously shown [6, 7] the unloaded sediment already contained 50 ppm Cu and no detectable Cd.
Electron Microscopy Cells were harvested by centrifuging in a clinical centrifuge and then fixed at room temperature for 1 h in 3% glutaraldehyde in 0.05M phosphate buffer at pH 7. I. The cells were then washed three times in buffer, postfixed for I h in cold 1% OsO 4, and then dehydrated in a graded acetone series. The dehydrated cells were
Effects of Metal Ions on Algal Ultrastructure
185
Table I. Distribution of different types of cells in cultures of Anabaena7120 treated with Cu and Cd
Treatment Untreated culture 10 -5 M Cu(NO3) 2 l0 -4 M Cu(NO3) 2 10-5 M Cd(NO3) 2 10-4 M Cd(NO3)2 Grown over Cu-loaded sediment Grown over Cd-loaded sediment
Cu or Cd Vegetative cells a bound (/ag/mg cells) b Normal Distorted
Heterocysts a Normal
Distorted
-7 N.D. 70 N.D. 0.004
92.5 89.0 1.6 3.2 0 90.4
0 2.6 20.9 84.9 82.7 0.7
7.5 4.2 5.4 0 1.8 6.7
0 4.2 72.0 11.9 15.5 2.2
0.058
27.3
61.2
1.4
10.1
a Figures show percent of each kind of cell. From 114 to 263 (usually ca. 130) cells were examined after each treatment. More details on the kinds of distortions observed are given in the text. bEstimated from the results of Laube et al. [7] or determined separately for these experiments as described in [7]: substantially higher amounts bound would be expected with the higher Cu or Cd concentrations, although these were not determined (N. D.).
infiltrated with Spurr's hard resin mixture [17], which was then polymerized at 60~ for 18 h. Silver- and gold-colored sections were cut with a DuPont diamond knife on a Sorval Porter-Blum MT-2B ultramicrotome, mounted on acetone-treated 200 mesh copper grids without support, and stained for 5 rain in uranyl acetate (5% in 50% ethanol) followed by 3 min in lead citrate [ 13]. Observations and photographs were made with a Philips 201 electron microscope.
Results and Discussion
Morphological Changes of Anabaena 7120 V e g e t a t i v e cells (Fig. 1) and heterocysts (Fig. 2) of Anabaena 7120 f r o m untreated cultures s h o w m o r p h o l o g i c a l features typical of this genus o f b l u e - g r e e n algae [14]. U n t r e a t e d cultures consisted entirely o f normal cells plus about 8% normal heterocysts (Table 1). C u l t u r e s treated with 10 -5 M C u ( N O 3 ) 2 s h o w e d little m o r p h o l o g i c a l change. W h e n the culture was treated with 1 0 - 4 M C u ( N O 3 ) 2, considerable lysis occurred, as s h o w n by the d e c r e a s e in turbidity and appearance o f blue p h y c o c y a n i n pigment in solution. This is r e f l e c t e d in the m u c h smaller proportion o f cells to heterocysts (Table 1). This must have resulted f r o m selective b r e a k d o w n o f vegetative cells with better preservation o f the t h i c k e r - w a l l e d heterocysts. H o w e v e r , m o s t heterocysts w e r e distorted, as were the r e m a i n i n g ceils. Cells with a corrugated appearance, mostly darkly stained (electron dense), w e r e the m o s t c o m m o n f o r m , although s o m e lighter corrugated cells of normal e l e c t r o n density w e r e also o b s e r v e d (Figs. 3 and 4). M o s t distorted heterocysts had a c o l l a p s e d appearance, and m a n y were empty. T h e degree to w h i c h heterocysts were c o l l a p s e d varied: s o m e w e r e indented or partly collapsed. Others were almost
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Fig. 1. Vegetative cell ofAnabaena 7120, untreated culture. Cg, cyanophycin granules; PB, polyhedral body; thylakoids (arrow). 22,500X. Fig. 2. Anabaena 7120 heterocyst, untreated culture. 18,000X.
Fig. 3. Anabaena 7120 vegetative cell grown over Cd-loaded sediment. Note the corrugated appearance of the cell. Is, intrathylakoidal space. 27,000X. Similar cells were seen after Cu 2+ addition to cultures. Fig, 4. Anabaena 7120 vegetative cell grown over Cd-loaded sediment. Note the much darker appearance of the cell compared with Fig. I. Is, intrathylakoidal space. 28,500X. Similar cells were seen after Cu 2+ or Cd 2+ addition to cultures.
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Fig, 5. Anabaena 7120 heterocyst. This cell had collapsed in such a way that in a cross sectionit appears hollow, Culture treated with 10-4M Cu 2+. 25,000X, Fig. 6, Anabaena 7120 heterocyst, showing partiallydisintegratedcell wall, Culturetreated with 10 -4M Cu 2+ . 21,000X. Fig. 7. Anabaena 7120 heterocyst, showingcompletelydisintegratedcell wall. Culturetreated with 10-4M Cu 2+, 21,400. Fig. 8. Anabaena 7120. A fieldof deformed cells. Culturetreated with 10 -SM Cd2+ . 4,200X.
completely flattened so that the cell contents were practically indistinguishable. That shown in Fig. 5 is so deeply indented that it appears hollow in cross section. In a number of half-empty heterocysts the cell walls had become networks of fine, electron-dense fibrils. That shown in Fig. 6 is partly fibriUar, whereas that in Fig. 7 is completely enclosed by a thick, diffuse layer of electron-dense fibers. It is uncertain if
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these fibers arise from the disintegrating cell wall or if they are synthesized as the wall breaks down. In contrast to Cu (NO3)2, Cd(NO3) 2 caused no visually observable lysis. The ratio of cells to heterocysts decreased only slightly (Table 1). Treatment with 10 -5 o r 10 -4 M Cd(NO3)2 distorted most or all the cells, leading mainly to the kind of highly electrondense, corrugated appearance shown in Fig. 4. An unusual type of shape distortion, found only in cells treated with 10 -5 M Cd(NO3) 2, is shown in Fig. 8. These cells were partly flattened in such a way that in sections at least one part of the cell appeared to have a straight edge. Most of the heterocysts were distorted; half-empty ones, as shown in Fig. 6, predominated. In cultures grown over Cu-loaded sediment, no significant change in the proportion of normal cells occurred. Some cells, however, had numerous intrathylakoidal spaces (Fig. 9), a feature rarely encountered in untreated cells. Centrally located empty spaces, which often contained polyhedral bodies, were observed in this experiment but never in untreated cultures. Polyhedral bodies were also more numerous than in untreated cells. Most heterocysts appeared normal, with only a small proportion of empty ones. In contrast, much more cell distortion was seen in cultures grown over Cd-loaded sediment (Table 1; Figs. 3 and 4), with corrugated cells, both highly electron dense and of normal electron density, predominating and roughly equal numbers of collapsed and empty heterocysts.
Morphological Changes in Ankistrodesmus braunii Figure 10 shows, in cross section, a young cell ofA. braunii from an untreated culture. The cell is still enclosed by the mother cell wail. Organelles characteristic of a chlorococcalean alga are seen: a chloroplast with a single pyrenoid; a nucleus with adjacent Golgi apparatus; and mitochondria, endoplasmic reticulum (ER), and ribosomes. In such untreated cultures, harvested in late exponential growth, about one-third of the cells appeared similar to that shown in Fig. 10, with all organelles clearly discernible. More than half the cells were more electron dense, but the large cell organelles---particularly the pyrenoid----could still be distinguished. The proportions of cell types remained roughly the same in cells grown in the presence of 10-5 Cu(NO3)2 (Table 2). When cells were grown in the presence of 10-4M Cu(NO3) 2, many were so electron dense that not even the pyrenoid was recognizable. The most striking result of treatment with high Cu 2+ concentration was the presence of multinucleate giant cells (Figs. 11-14). These cells presumably resulted from repeated mitotic division without subsequent cytokinesis. Some, however, were undergoing cytokinesis (Fig. 11). We could not determine what fraction of such cells finally undergo cytokinesis or remain multinucleate. In fact, most giant cells were so electron dense that it was difficult to know what division processes had occurred. In addition to the multinucleate giant cells, other giant cells had several daughter cells, probably in multiples of four, still enclosed by a mother cell wall. Figure 15 shows an electron-dense cell containing at least 12 daughter cells. The walls of giant cells were considerably thicker than those of normal mother cells. An unusual feature of some giant cells, never seen in untreated cultures, consisted of three parallel strands of endoplasmic reticulum connected to the nuclear envelope and traversing at least half the cell length (Fig. 12). A section of a nucleus with a number of
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Fig. 9. Anabaena 7120 vegetative cell grown over Cu-loaded sediment. PB, polyhedral body; Is intrathylakoidal space. 32,000X. Fig. 10. Ankistrodesmus braunii, untreated culture showing the main cell organelles. N, nucleus; G, Golgi apparatus; P, pyrenoid;m, mitochondrion.30,500X. deep indentations is shown in Fig. 13. Such indentations were also found only in giant cells, and it is uncertain if such nuclei can subsequently divide. Pickett-Heaps [11] suggests that in the genus Ankistrodesmus centrioles are transitory structures, appearing only during mitosis and cell cleavage. We have found centrioles in the giant cells of A. braunii. Two of them, partly enclosed by the nucleus, are seen in Fig. 14.
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Fig. 11, Ankistrodesmus braunii, A giant cell showing 5 nuclei (N). The cell is undergoing cytokinesis as indicated by cleavage furrows (arrows). Culture treated with 10 -4M Cu 2 + 10,000X, Fig. 12. Ankistrodesmus braunii. Giant cells, showing part of the nucleus (N) and 3 parallel strands of endoplasmic reticulum (ER) connected with the nuclear envelope (arrows). Culture treated with 10-4M Cu 2+. 27,000X. Fig. 13. Ankistrodesmus braunii. A large nucleus (N) of a giant cell showing 2 deep indentations which in a cross section appear hollow. Culture treated with 10 -4M Cu 2+ . 26,000X. Fig. 14. Ankistrodesmus braunii giant cell showing 2 centrioles (arrows) adjacent to the nucleus (N). Note the thick cell wall (CW). Culture treated with t0 -4M Cu 2+. 49,500X.
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Table 2. Distributionof differenttypes of cells in culturesofA. brauniitreated withCu and Cd
Treatment Untreatedculture 10 -5 M Cu(NO3)2 10 -4 M Cu(NO3)2 10-5 M Cd(NO3)2 Grown over unloded sedimentc Grown over Cu-loaded sediment Grown over Cd-loaded sediment
Cu or Cd bound (tag/mg cells)b
Normal cells a
Very electron dense cellsa
Giant cellsa
Dead ceUsa
-0.7 3.0 1.4 0.016
90.2 95.4 3.5 0 95.7
0 0 61.5 96.5 0
0 0 26.5 0 0
9.8 4.6 8.4 3.5 4.3
0.04
98.2
0
0
1.8
0.025
93.7
0
0
6.3
aFigures show percentof each kind of cell. From 142 to 329 (usuallyca. 220) cells were examinedafter each treatment. Both more- and less-electron-densecells are includedin the countsof normaland giant cells (see text). Cells partlyor entirelyemptyof internalcontentsare considered"dead." Veryelectron-densecellswere so dark that no internalstructureswere visible. bFrom the resultsof Laubeet al. [7] or determinedseparatelyfor these experiments. cThis sedimentalreadycontained50 ppm Cu [6].
After growth in the presence of 10-5 M Cd(NO3)2, practically all (96.5%) of the cells were very electron dense, with virtually indistinguishable cell organelles (Fig. 16). No giant cells were found. As previously pointed out [7], the presence of Cu- or Cd-loaded sediment did not inhibit the growth o f A . braunii, even though some of the metals became bound to the algal cells. No significant change of morphology was seen after growth in the presence of sediment (Table 2); cells that were electron dense still had discernible organelles. Although the sites of Cu and Cd action on both species are not known, our results suggest that they may act on the outer cell layers. The lysis of vegetative cells of Anabaena 7120 by Cu 2+ may reflect an action on the cell walls. The corrugated appearance caused by Cu 2+ and Cd 2+ may be due to a direct action on the walls, o r t o an osmotic effect of the f'Lxative on cells with weakened walls. The unusual distortion of cells exposed to 10-5 M Cd (Fig. 8) could also be due to an effect on wall rigidity that causes cells to flatten against each other during centrifugation. The inhibition by Cu 2+ of cytokinesis and/or cell separation in A. braunii might also reflect an action o f this metal on the cell's outer surface. Cu 2+ can inhibit cell division in Chlorella pyrenoidosa [ 19] and C. ellipsoidea [4], and can cause giant cell formation in C. vulgaris [3]. Mercury can also cause giant cell formation in a Ch'lorella sp. and the green flagellate Dunaliella tertiolecta [2]. It might be profitable for further studies of such metals to concentrate on the cell surface as a site of action.
Acknowledgments. This work was supported by grants from environmentCanada and The Natural Sciences and EngineeringResearch Councilof Canada.
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Fig. 15. Ankistrodesmus braunii dark giant cell showing at least 12 daughter cells enclosed by a mother cell wall. Culture treated with 10 - 4 M Cu 2+. 1 I, 100X. Fig. 16. Ankistrodesmus braunii dark cell. Note high electron opacity of the cell and lack of morphological details. Culture treated with 10 - 5 M Cd 2+ . 20,000X.
References I. Bartlett, L., F. W. Rabe, and W. H. Funk: Effects of copper, zinc and cadmium on Selenastrum capricornutum. Water Res. 8, 179-185 (1974) 2. Davies, A. G.: An assessment of the basis of mercury tolerance in Dunaliella tertiolecta. J. Mar. Biol. Assoc. U.K. 56, 39-57 (1976) 3. Foster, P. L.: Copper exclusion as a mechanism of heavy metal tolerance in a green alga. Nature 269, 322-323 (1977) 4. Kanazawa, T., and K. Kanazawa: Specific inhibitory effect of copper on cellular division in Chlorella. Plant Cell Physiol. 10, 495-502 (1960) 5. Klass, E., D. W. Rowe, and E. J. Massaro: The effect of cadmium on population growth of the green alga Scenedesmus quadricauda. Bull. Environ. Contam. Toxicol. 12, 442-445 (1974) 6. Laube, V. M., S. Ramamoorthy, D. J. Kushner: Mobilization and accumulation of sediment-bound heavy metals by algae. Bull. Environ. Contam. Toxicol. 21,763-770 (1979) 7. Laube, V. M., C. McKenzie, and D. J. Kushner: Strategies of algal dealings with heavy metals. Can. J. Microbiol. 26, 1300-1311 (1980) 8. Nuzzi, R.: Toxicity of mercury to phytoplankton. Nature 237, 38--40 (1972) 9. Ohad, I., P. Siekevitz, and G. E. Palade: Biogenesis of chloroplast membranes. J. Cell Biol. 35, 521-552 (1967) 10. Overnell, J.: The effect of some heavy metal ions on photosynthesis in a freshwater alga. Pesticide Biochem. Physiol. 5, 19-26 (1975) 11. Pickett-Heaps, J. D.: Green Algae--Structure, Reproduction and Evolution in Selected Genera. Sinaur Associates, Publishers, Sunderland, MA (1975)
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12. Ramamoorthy, S., A. Massalski, and T. Bistricki: Energy dispersive X-ray microanalysis of mercury in environmental samples. Proceedings of the Symposium on Electron Microscopy and X-ray Applications to Environmental and Occupational Health Analyses, Vol. I, pp. 247-254. Ann Arbor Sciences Publishers, Ann Arbor (1978) 13. Reynolds, E. S.: The use of lead citrate at high pH as an electron-opaque stain in bacteria. J. Bacteriol. 70, 691-701 (1963) 14. Shively, J. M.: Inclusion bodies in prokaryotes. Annu. Rev. Microbiol. 28, 167-187 (1974) 15. Silverberg, B. A.: Ultrastructural localization of lead in Stigecloniam tenue (Chlorophyceae, Ulotrichales) as demonstrated by cytochemical and X-ray microanalysis. Phycologia 14, 265-274 (1975) 16. Silverberg, B. A.: Cadmium-induced ultrastructural changes in mitochondria of freshwater green algae. Phycologia 15, 155-159 (1976) 17. Spurr, A. R.: A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) 18. Stanier, R. Y., R. Kunisawa, M. Mandel, and G. Cohen-Bazire: Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 31, 171-205 ( 1971) 19. Steeman Nielsen, E., and L. Kamp-Nielsen: Influence of deleterious concentrations of copper on the growth of Chlorellapyrenoidosa. Physiol. Plant. 23, 828-840 (1970) 20. Steeman Nielsen, E., and S. Wium-Anderson: The influence of Cu on photosynthesis and growth in diatoms. Physiol. Plant. 24, 480--484 (1971)
