Folia Microbiol.

49 (5), 549556 (2004)

http://www.biomed.cas.cz/mbu/folia/

Zoosporangia Survival, Dehiscence and Zoospore Formation, and Motility in the Green Alga Rhizoclonium hieroglyphicum As Affected by Different Factors
S. GUPTA, S.C. AGRAWAL
Department of Botany, University of Allahabad, Allahabad, India Received 14 October 2003 Revised version 8 March 2004

ABSTRACT. Urea at 200 ppm (probably serving as a nitrogen source), liquid Bolds basal medium at pH 7.5, temperature of about 22 C and light intensity of about 40 mol m2 s1 for 16 h a day induced rapid and/or abundant zoospores formation and zoosporangia dehiscence and favored zoospore liberation, speed and motility time period in the green alga Rhizoclonium hieroglyphicum. However, factors such as water stress (2 and 4 % agarized media, liquid media with 0.20.4 mol/L NaCl, 560 min blot-dryness of filaments), pH extremes of liquid media (at 6.5 and 9.5), temperature shock in liquid media (5 and 35 C for 5 min), UV exposure (0.963.84 kJ/m2), lack of all nutrients from liquid medium (double distilled water), darkness, and presence of heavy metals (125 ppm Cu, Fe, Zn, Hg, Ni, Co) or organic substances (200 600 ppm captan or DDT, 800 and 1000 ppm 2,4-D, 50 and 400 ppm indole-3-acetic acid (3-IAA), 1000 and 2000 ppm urea, 100 and 200 ppm thiourea) in liquid media decreased and/or delayed at various levels either zoosporangia survival, zoospore formation or zoosporangia dehiscence and/or the rate of zoospore liberation from zoosporangia, zoospore speed and time period of motility in the media or totally inhibited all these processes. 3-IAA at 50 and 400 ppm induced zoosporangial papilla to grow into a tube-like projection of about 30120 m in length. Zoosporangial dehiscence rather than zoospore formation or zoosporangia survival, and zoospore motility period rather than zoospore speed are probably more sensitive to various adverse environmental factors. The rate of zoospores liberation from zoosporangium (possibly related directly to some extent on the zoospore number inside) is probably independent of zoospore speed in the medium.

Information on the effects of various environmental factors such as water stress (Klebs 1896; Bold 1933; Starr 1954a,b), light (Neeb 1952; Lewin 1956; Mason 1965; Kulfinski and Henschel 1969; Coss and Pickett-Heaps 1973), temperature (Stewart and OKelley 1966; Hoffmann and Graham 1984; Graham et al. 1985, 1986) and pH (Agrawal 1993) on zoospore formation in some green algae is relatively scarce and often not recent. UV light was found to affect the survival rate in some brown algal zoospores (Wiencke et al. 2000; Swanson and Druehl 2000). Since pollutants in water may cause changes in the swimming performance of zoospores or flagellates, the movement of the flagellate Euglena gracilis has been developed as the Ecotox test to act as early warning system to detect deterioration in water quality (Tahedl and Hder 1999). Here we demonstrate the effects of water stress, pH variation, temperature shock, UV exposure, lack of all nutrients from the medium, darkness and addition of heavy metals (Cu, Fe, Zn, Hg, Ni, Co) or organic substances (captan; DDT; 2,4-D; indole-3-acetic acid, 3-IAA; urea; thiourea) on zoosporangia survival, zoospore formation, zoosporangia dehiscence, zoospore speed and time period of motility in Rhizoclonium hieroglyphicum (C. AGARDH) KTZING.

MATERIAL AND METHODS Algal material and growth conditions. R. hieroglyphicum was collected while growing firmly attached to cement walls in a running freshwater shallow tank on the university campus (University of Allahabad, India). Clonal cultures were raised through germinating zoospores and were maintained under controled culture conditions in Bolds basal medium (BM) (Nichols and Bold 1965; pH adjusted prior to autoclaving to 7.5) at c. 22 C and light intensity of c. 40 mol m2 s1 from daylight fluorescent tubes for 16 h a day. Ten-d-old filaments were mature vegetative ones (Fig. 1A). Filaments of vegetative cells started zoosporangia differentiation, as evident by an increase in their breadth up to 1.5 times and change in color from light to dark green, in 1520-d-old cultures. (Fig. 1B,C). Twenty-d-old zoosporangial filaments were used as a source of inoculum for all experiments (hereafter called algal material; zoosporangia and zoospore

550 S. GUPTA and S.C. AGRAWAL

Vol. 49

Fig. 1. A: R. hieroglyphicum 10-d-old mature vegetative filaments; 100; B: a 15-d-old filament showing initiation of zoosporangia formation; 100; C: a 20-d-old zoosporangial filament; 100; D: zoosporangium showing a papilla and zoospore primordia; 600; E: a zoosporangial filament showing a zoosporangium with mature zoospores and an another with zoospore primordia; 200; F: a zoosporangium with mature zoospores; 200; G: released zoospores motile in the medium; 400; H: zoospores settled to substratum; 400; I: a dead zoosporangium releasing its cytoplasmic content on 4 % agarized medium; 400.

2004

ZOOSPORANGIA AND ZOOSPORE FORMATION IN R. hieroglyphicum 551

formation in 10- and 20-d-old filaments occurred more prolifically when they were transferred to fresh BM than in old parent BM, indicating that fresh nutrient supply is important for them). Zoosporangium-initiated zoospore formation by dividing its content into a large number of primordia. A cell-wall papilla developed during zoospore primordia formation (Fig. 1D). After formation, mature zoospores moved rapidly inside the zoosporangium about 30 min prior to escaping into the medium, through a lateral pore situated either on a papilla or anywhere on the wall, one by one (Fig. 1E,F). Released zoospores were yellowish-green and their diameter achieved on average 3.25 m (Fig. 1G). Most of the zoospores of a zoosporangium escape into the medium, continuously or intermittently, within 34 min of the process start, but some (even up to 20 %) did not leave and lay quiescent after moving for about 30 min inside it. Quiescent zoospores remaining in the zoosporangium died within 23 d and thereafter disintegrated. The released zoospores swam in the medium for 30120 min or more (Fig. 1G) and then settled (Fig. 1H) to cell walls of the parent filaments and walls of the flask. Water stress. Algal material obtained from liquid BM was exposed to water stress either by spreading it on solid BM containing 2 or 4 % agar, or inoculating it in liquid BM with 0.20.4 mol/L NaCl. Another part of the algal material was completely blot-dried for 560 min and then inoculated into liquid BM and maintained in the culture chamber. pH. The algal material was inoculated into liquid BM adjusted to pH 510.5 (determined with Systronic digital pH meter) prior to autoclaving by adding 1 mol/L HCl or 2 % NaOH and then kept in the culture chamber. Temperature shock. Algal material suspended in BM was placed at 550 C; aliquots were withdrawn after 560 min and then inoculated into fresh BM and maintained in the culture chamber. UV exposure. After placing in 10 mL sterile distilled water, the material was spread in open Petri dishes (diameter 90 mm) and exposed to UV light from a Philips germicidal lamp (main output at 254 nm and fluence rate of 3.2 W/m2). The energy fluence of UV light which was obtained by increasing the time of exposure from 5 to 20 min ranged from 0.96 to 3.84 kJ/ m2. After irradiation, the material was centrifuged, transferred to fresh BM and maintained in the culture chamber. Lack of all nutrients. Algal material inoculated in glass double-distilled water (pH adjusted prior to autoclaving to 7.5) was kept in the culture chamber. Acid-washed Borosil glassware properly rinsed with double distilled water was used. Darkness. Culture tubes containing inoculated algal material in liquid BM were wrapped in black paper and kept in the dark (no light as measured using a luxmeter; Lutron, USA) in the culture chamber. Metals (Cu, Fe, Zn, Hg, Ni, Co) added as copper sulfate (99 %; Merck, India), ferrous sulfate (99 %), zinc sulfate (99 %), mercuric dichloride (98 %; all three SD Fine Comp., India), nickel sulfate (99 %; Merck, India), or cobalt dinitrate (97 %; BDH, India) were dissolved and individually mixed with BM (pH adjusted prior to autoclaving to 7.5) to a final concentration of 125 ppm. Inoculated culture tubes were maintained in the culture chamber. Organic substances. Captan (50 %; Rallis, India), DDT (10 %; Governmental supply, India), 2,4-D (86 %; Tropical Agro, India), urea (nitrogen 46 %; Vijaypur Fertilizer Comp., India) or thiourea (99 %; Merck, India) were added to the BM (pH adjusted prior to autoclaving to 7.5) to a final concentrations of 1002000 ppm. 3-IAA (100 %; Loba-Chemie, India) was dissolved separately in a minimal volume of 80 % ethanol (23 drops) and mixed slowly into a known amount of BM to prepare the hormone solution of desired concentrations of 50400 ppm. The pH was adjusted prior to autoclaving to 7.5. A separate control was maintained for IAA treatment containing the same amount of ethanol as in the IAA solution. All inoculated culture tubes were placed in the culture chamber. All algal material was observed to assess zoosporangia survival, dehiscence, zoospore number in zoosporangia, rate of zoospore liberation from zoosporangia, zoospore speed and motility retention on 10 d of inoculation except as otherwise indicated. Zoosporangia survival. Percentage of zoosporangia survivability was determined by counting the number of living zoosporangia with respect to dead zoosporangia (looking hyaline, pale-yellowish or brownish in color) out of about 80009000 zoosporangia of the filaments counted from each of 3 replicates. Zoosporangia dehiscence. Percentage of dehisced zoosporangia, showing release of zoospores, was determined with respect to all surviving zoosporangia. Possible zoospore number in zoosporangia. Counting was done of zoospores when they escape into the medium from most of the zoosporangia. A zoosporangium may contain up to 800980 zoospores. Rate of zoospore release from zoosporangia. Out of total zoospores present in zoosporangia, how many escaped into the medium in 1 min was determined. Zoospore speed and motility retention in the medium. Media after release of zoospores (Fig. 1G) were placed on a slide underneath the coverslip and viewed through a calibrated ocular micrometer in a microscope.

552 S. GUPTA and S.C. AGRAWAL

Vol. 49

Zoospore speed was determined by following their path of movement (which was very irregular) with respect to microscale and was expressed in m/min. About 1000 zoospores were assessed for each of three replicates in each case. In control algal material the zoospore speed range was between 120 and 200 m/min. How long the zoospores retain motility underneath the coverslip was also determined. Since most of them (even 80 %) retain motility for 2 h or more under the same conditions, an additional amount of the same medium (centrifuged one and having no zoospore) was added periodically dropwise underneath the cover slip, so that it should not dry and remain filled with the medium.

RESULTS AND DISCUSSION Water stress. Although most of R. hieroglyphicum zoosporangia survived and formed zoospore primordia, none dehisced on 2 or 4 % agarized BM even on 30 d after inoculation (Table I). However, about 2 % dead zoosporangia slowly released their cytoplasmic content on 4 % agarized BM 10 d after inoculation (Fig. 1I). The presence of water was required for development of zoospores in Hormidium sp. and Vaucheria sp. (Klebs 1896), gametes in Protosiphon sp. (Bold 1933), and zoospores in Plaktosphaeria sp. and Tetraedron sp. (Starr 1954a,b). Water stress inhibited zoosporangia formation and zoospore germination in Cladophora glomerata and R. hieroglyphicum (Agrawal and Singh 1999), autospore release in Chlorella vulgaris (Agrawal and Singh 2001) and formation of heterocysts in Nostoc calcicola and Scytonema hofmanni, oogonium in Oedogonium sp. and daughter net in Hydrodictyon reticulatum (Agrawal and Singh 2002; Agrawal and Pal 2003).
Table I. Effects of water stress, pH, temperature shock, UV exposure, lack of all nutrients, darkness, heavy metals, and organic substances on zoosporangia survivability (ZOS), zoosporangia dehiscence (ZOD), possible zoospore number in zoosporangia (PZN), rate of zoospore release from zoosporangia (RZR), zoospore speed (ZS), and zoospore motility retention (ZMR) in R. hieroglyphicuma Condition/treatment ZOS ZOD Controlc Liquid BM 100 22 Water Agarized BM, % 2 4 NaCl in liquid BM, mol/L 0.2 0.3 0.4 Blot-dryness of filaments, min 5d 10e 20 60 30 20 19 13.5 pH 5.0 6.5e 7.5 9.5 10.5e 45 62 100 92.5 75 Temperature Temperature, C 5 5 5 min 5e 10e 30 of 2.5 1.5 0 0 liquid 0 7.5 24 6 1 shock 800900 600800 0 0 BM 0 800900 800920 800900 800900 in liquid 2022 4045 2025 0310 BM 120200 120200 066110 084120 0 10130 30150 05100 05100 1520 0570 096160 054900 100 05100 0 0 88 79 70 12 2 0 800900 450500 ZF 4045 0510 0 120200 080140 30900 100 0 92 90 0 0 ZF ZF 0 0 0 0 800950 stress 3545 120200 30150 PZNb RZR ZS ZMR0

96 85 51

0.5 0 0

500600 ZF 0

20 0

108180

30450 0 0

2004

ZOOSPORANGIA AND ZOOSPORE FORMATION IN R. hieroglyphicum 553

15 35 35 40 50

60 5 60 5 5

100 73 54 25 1 UV

21 0 0 0 0

800950 ZF ZF 0 0

3240 0 0

120200

30900 0 0 0 0

exposure

Dose, kJ/m2 0.96d 1.92 3.84 96 85 50 Lack Double-distilled water 10 of 2 0 0 all 0 Darkness Zero light intensity 97 Metals Copper 1e 5 25 Iron 1e 5 25 Zinc 1e 5 25 Mercury 1 5 25 Nickel 1 5 25 Cobalt 1e 5 25 Organic Captan 200 400 600 DDT 200 400 600 45 30 16 0 0 0 0 0 0 0 0 0 continued 82.5 61 50 1 0 0 400500 0 0 0580 080130 50 0 0 92 83 74 1 0 0 substances in 250300 ZF 0 liquid 0510 0 BM, ppm 030450 50 0 0 92 77.5 65 8 3.5 0 600750 400550 ZF 1020 0510 0 120160 084140 300 100 0 35 6.5 0 0 0 0 0 0 0 0 92 80 67 1.5 0 0 300400 ZF ZF 810 0 0 040500 50 0 0 98 90 78 1 0 0 250400 ZF ZF 0510 0 0 036600 50 0 0 96 90 73 2.5 0 0 300450 ZF ZF 1015 0 0 042700 05100 0 0 in 0 liquid ZF BM, ppm 0 0 800950 ZF 0 nutrients 0 0 3545 0 120200 30900 0 0

554 S. GUPTA and S.C. AGRAWAL

Vol. 49

2,4-D 200 800d 1000 3IAA 0 50 400 Urea 200g 400g 1000 2000e Thiourea 100 200 14 5 0 0 0 0 0 0 100 100 100 100 50 36 10 2 950980 800860 450550 250430 5060 3035 2532 10 120200 120200 108180 060100 90150 15300 15200 5 100 100 100 20 0f 0f 900950 ZF ZF 3745 0 0 120200 30900 0 0 100 90 84 10 2.5 0 950980 400550 ZF 3545 25 0 120200 108180 30120 10200 0

aZOS (in %), ZOD (% with respect to ZOS), RZR (% of zoospores released with respect to total present per min), ZS (in m/min),

ZMR (in min); all readings on 10 d after inoculation except otherwise indicated; rounded mean of 3 replicates; for details see Material and Methods. bZF represent that zoospores primordia are formed, while 0 that they are not, even 30 d after inoculation. cAlgal material inoculated in fresh liquid BM (pH adjusted prior to autoclaving to 7.5) and maintained as usual in culture chamber at c. 22 C and light intensity of 40 mol m2 s1 for 16 h a day. d,eAll readings on 20, 30 d after inoculation, respectively, since zoospores formation and zoosporangia dehiscence were delayed as compared to the control. fAbout 15, 40 % zoosporangial papillae grew into a tube-like projection of about 30 and 120 m length in 50 and 400 ppm 3-IAA, respectively. gAll readings 6 d after inoculation since zoospore formation and zoosporangia dehiscence occurred earlier and/or more prolific than in the control.

R. hieroglyphicum zoosporangial dehiscence was affected in 0.2 mol/L NaCl but other stages also in 0.3 mol/L NaCl. No zoosporangia dehisced in 0.4 mol/L NaCl. In saline streams, Round (1964) found immobile forms of diatom species which normally were mobile. Blot-dryness of algal material for even 5 min dramatically decreased zoosporangial survival and dehiscence. No zoospore formation occurred in a 20-min blot-dried algal material. pH. All zoosporangial stages studied (i.e. zoosporangia survival, dehiscence, zoospore number in zoosporangia, rate of release, speed and motility retention) favored pH 7.5; most of them were affected adversely as the pH decreased to 6.5 or increased to 10.5. No zoospore formation and zoosporangia dehiscence occurred at pH 5. Burkholder (1933) observed that the range of pH of 6.49.5 was most favorable to the movement of Oscillatoria formosa. In the green alga Stigeoclonium pascheri, zoospore formation was maximum at pH 8 and decreased below pH 6 (Agrawal 1993). For zoosporangia formation and zoospore germination in C. glomerata and R. hieroglyphicum pH of 78 was optimum (Agrawal and Misra 2002). Temperature shock. Exposure of algal material to 5 C for even 5 min adversely affected almost all zoosporangial stages beside zoosporangia survivability, while exposure to 35 C for the same time stopped all zoosporangia dehiscence. Control algal material growing at about 22 C exhibited all zoosporangial stages favorably. (R. hieroglyphicum collected from nature exhibited all zoosporangial stages during mid-February to mid-March and mid-October to mid-November when the water temperature was about 2025 C. The alga exists only in the vegetative stage and did not show any zoosporangial stage during JulyAugust when the water temperature was 3031 C. However, no zoosporangia present in the alga dehisced during mid-December to January when the water temperature was 614 C.) Optimum temperature for zoosporogenesis in Cladophora sp. was between 15 and 20 C (Mason 1965), in Protosiphon botryoides between 20 and 30 C (Stewart and OKelley 1966), in Ulothrix zonata near 20 C (Graham et al. 1985) and in Coleochaete scutata at 2025 C (Graham et al. 1986). Zoosporogenesis in green algae C. glomerata (Hoffmann and Graham 1984) and C. scutata (Graham et al. 1986) declined above 30 C. UV exposure. Zoosporangial dehiscence was affected at 0.96 kJ/m2 of UV treatment and did not occur at all at and beyond 1.92 kJ/m2 of UV. UV light inhibited motility and phototaxis in Euglena gracilis (Hder and Hder 1988), survival rate in some brown algal zoospores (Wiencke et al. 2000; Swanson and

2004

ZOOSPORANGIA AND ZOOSPORE FORMATION IN R. hieroglyphicum 555

Druehl 2000) and cell division in Chroococcus sp., Gloeocapsa sp. and Aphanothece sp., formation of heterocysts in Scytonema hofmanni, oogonia in Oedogonium sp., and daughter nets in Hydrodictyon sp. (Agrawal and Pal 2003); it also induced oxidative damage in Chlorella pyrenoidosa (Chen et al. 2003). Lack of all nutrients. Most of R. hieroglyphicum zoosporangia died without forming any zoospores when kept in double-distilled water indicating that nutrients present in BM were essential for zoosporangia survival and zoospore formation. Low levels or lack of nitrogen or phosphorus in culture media decreased zoospore formation in P. botryoides (OKelley and Deason 1962), Botrydiopsis sp., Bumilleriopsis sp. and Pseudollumilleriopsis sp. (Pecora and Russell 1973) and Chlorococcum echinozygotum (OKelley 1984) and zoosporangia formation and zoospore germination in C. glomerata and R. hieroglyphicum (Agrawal and Misra 2002). Darkness. Although R. hieroglyphicum zoosporangia survived and formed zoospores primordia, none dehisced even after 30 d of inoculation in darkness. Presence of light is needed for formation of zoospores in H. reticulatum (Neeb 1952), Enteromorpha sp. (Lersten and Voth 1960), Cladophora sp. (Mason 1965) and in Schizomeris sp. (Kulfinski and Henschel 1969) and akinetes in some blue-green algae and Pithophora oedogonia (Agrawal and Singh 2000). Metals. Copper, iron, mercury, nickel and cobalt, all at 125 ppm, variously suppressed the survival of zoosporangia and considerably or completely inhibited the zoospore formation, zoosporangia dehiscence, rate of zoospore liberation, zoospore speed and motility time period. Growth inhibition in algae after exposure to metals has been attributed to inhibition of function of photosynthetic pigments, to enzyme inhibition, uptake of nutrients or damage to cell membrane (Stokes 1983; De Filippis and Pallaghy 1994; Fathi 2002). Organic substances. Thiourea at 100 and 200 ppm, DDT at 200600 ppm, and captan at 400 600 ppm affected zoosporangia survival and completely inhibited zoosporangia dehiscence. Captan at 200 ppm reduced most of zoosporangial stages to a very low level. 2,4-D at 8001000 ppm decreased or altogether inhibited zoosporangia dehiscence. Although all zoosporangia survived and formed zoospores, none dehisced at 50 and 400 ppm of 3-IAA. However, about 15 and 40 % zoosporangial papillae grew into a tube-like structure of about 30 and 120 m length in 10-d treatment with 50 and 400 ppm 3-IAA, respectively. IAA at certain levels favored formation of zoospores in Ulothrix sp. (Conrad et al. 1959). Urea at 200 ppm (probably serving as nitrogen source) induced rapid and prolific dehiscence of zoosporangia and led zoospores to move longer in the medium, but at 1000 and 2000 ppm levels, it inhibited both processes. Various experiments done in our study suggest that different zoosporangial stages of R. hieroglyphicum occurred optimally in the presence of urea at 200 ppm, in liquid BM at pH 7.5, at about 22 C and at light intensity of about 40 mol m2 s1 for 16 h a day. Any alteration in the physical and chemical environment suppressed them variously. It is also evident that zoosporangia dehiscence rather than zoospore formation or zoosporangia survival, and zoospore motility time period rather than speed are more sensitive to various adverse environmental factors. The rate of zoospore liberation from zoosporangium (may be related directly to some extent to the zoospore number inside) is probably independent of zoospore speed in the medium.
REFERENCES AGRAWAL S.C.: Effects of some factors on the zoospore formation in Stigeoclonium pascheri (VISCHER) COX and BOLD. Phykos 32, 4749 (1993). AGRAWAL S.C., MISRA U.: Vegetative survival, akinete and zoosporangium formation and germination in some selected algae as affectted by nutrients, pH, metals, and pesticides. Folia Microbiol. 47, 527534 (2002). AGRAWAL S.C., PAL U.: Viability of dried vegetative cells or filaments, survivability and/or reproduction under water and light stress, and following heat and UV exposure in some blue-green and green algae. Folia Microbiol. 48, 501509 (2003). AGRAWAL S.C., SINGH V.: Viability of dried vegetative cells and the formation and germination of reproductive structures in Pithophora oedogonia, Cladophora glomerata and Rhizoclonium hieroglyphicum under water stress. Folia Microbiol. 44, 6370 (1999). AGRAWAL S.C., SINGH V.: Vegetative survival, akinete formation and germination in three blue-green algae and one green alga in relation to light intensity, temperature, heat shock and UV exposure. Folia Microbiol. 45, 439446 (2000). AGRAWAL S.C., SINGH V.: Viability of dried cells, and survivability and reproduction under water stress, low light, heat, and UV exposure in Chlorella vulgaris. Israel J.Plant Sci. 49, 2732 (2001). AGRAWAL S.C., SINGH V.: Viability of dried filaments, survivability and reproduction under water stress, survivability following heat and UV exposure in Lyngbya martensiana, Oscillatoria agardhii, Nostoc calcicola, Hormidium fluitans, Spirogyra sp. and Vaucheria geminata. Folia Microbiol. 47, 6167 (2002). BOLD H.C.: The life history and cytology of Protosiphon botryoides. Bull.Torrey Bot.Club 60, 241300 (1933). BURKHOLDER P.R.: Movement in the Cyanophyceae. The effect of pH upon movement in Oscillatoria. J.Gen.Physiol. 16, 875881 (1933).

556 S. GUPTA and S.C. AGRAWAL

Vol. 49

CHEN K., FENG H., ZHANG M., WANG X.: Nitric oxide alleviates oxidative damage in the green alga Chlorella pyrenoidosa caused by UV-B radiation. Folia Microbiol. 48, 389393 (2003). CONRAD H., SALTMAN P., EPPLEY R.: Effects of auxin and gibberellic acid on growth of Ulothrix. Nature 184, 556557 (1959). COSS R.A., PICKETT-HEAPS J.D.: Gametogenesis in the green alga Oedogonium cardiacum. I. The cell division leading to the formation of spermatids and oogonia. Protoplasma 78, 2139 (1973). DE FILIPPIS L.F., PALLAGHY C.K.: Heavy metals: sources and biological effects, pp. 3177 in L.C. Rai, J.P. Gaur, C.J. Soeder (Eds): Algae and Water Pollution. Schweizerbartsche Verlagsbuchhandlung, Stuttgart (Germany) 1994. FATHI A.A.: Toxicological response of the green alga Scenedesmus bijuga to mercury and lead. Folia Microbiol. 47, 667671 (2002). GRAHAM J.M., GRAHAM L.E., KRANZFELDER J.A.: Light, temperature and photoperiod as factors controlling reproduction in Ulothrix zonata (Ulvophyceae). J.Phycol. 21, 235239 (1985). GRAHAM L.E., GRAHAM J.M., KRANZFELDER J.A.: Irradiance, daylength and temperature effect on zoosporogenesis in Coleochaete scutata (Chlorophyceae). J.Phycol. 22, 3539 (1986). HDER D.-P., HDER M.A.: Inhibition of motility and phototaxis in the green flagellate Euglena gracilis by UV-B radiation. Arch. Microbiol. 150, 2025 (1988). HOFFMANN J.P., GRAHAM L.E.: Effects of selected physicochemical factors on growth and zoosporogenesis of Cladophora glomerata (Chlorophyta). J.Phycol. 20, 17 (1984). KLEBS G.: Die Bedingungen der Fortpflanzung bei einigen Algen und Pilzen. Jena Ed. 2, 1543 (1896). KULFINSKI F.B., HENSCHEL F.R.: The asexual reproduction of Schizomeris and Ulothrix (Chlorophyta) in response to various light intensities. Trans.Illinois Acad.Sci. 62, 303307 (1969). LERSTEN N.R., VOTH P.D.: Experimental control of zooid discharge and rhizoid formation in the green alga Enteromorpha. Bot.Gaz. 122, 3345 (1960). LEWIN R.A.: Control of sexual activity in Chlamydomonas by light. J.Gen.Microbiol. 15, 170185 (1956). MASON C.P.: Ecology of Cladophora in farm ponds. Ecology 46, 421428 (1965). NEEB O.: Hydrodictyon als Object einer vegleichenden Untersuchung physiologische BroBen. Flora (Jena) 139, 3995 (1952). NICHOLS H.W., BOLD H.C.: Trichosarcina polymorpha gen. et sp.nov. J.Phycol. 1, 3438 (1965). OKELLEY J.C.: Nitrogen and gamete production in Chlorococcum echinozygotum, Chlorophyceae. J.Phycol. 20, 220225 (1984). OKELLEY J.C., DEASON T.R.: Effect of nitrogen, sulfur and other factors on zoospore production by Protosiphon botryoides. Am.J.Bot. 49, 771777 (1962). PECORA R.A., RUSSELL C.R.: Zoospore production in selected Xanthophycean algae. Brit.Phycol.J. 8, 321324 (1973). ROUND F.E.: The ecology of benthic algae, pp. 138184 in D.F. Jackson (Ed.): Algae and Man. Plenum Press, New York 1964. STARR R.C.: Reproduction by zoospores in Planktosphaeria gelatinosa G.M. SMITH. Hydrobiology 6, 392397 (1954a). STARR R.C.: Reproduction by zoospores in Tetraedron bitridens. Am.J.Bot. 41, 1720 (1954b). STEWART J.R., OKELLEY J.C.: Periodic zoospore production by Protosiphon botryoides under alternating lightdark periods. Am.J.Bot. 53, 772777 (1966). STOKES P.M.: Response of freshwater algae to metals, pp. 87111 in F.E. Round, V.J. Chapman (Eds): Progress in Phycological Research, Vol. 2. Elsevier Science Publisher, Amsterdam (The Netherlands) 1983. SWANSON A.K., DRUEHL L.D.: Differential meiospore size and tolerance of ultraviolet light stress within and among kelp species along a depth gradient. Marine Biol. 136, 657664 (2000). TAHEDL H., HDER D.-P.: Fast estimation of water quality using the automatic biotest Ecotox based on the movement behavior of a freshwater flagellate. Water Res. 33, 426432 (1999). WIENCKE C., GOMEZ I., PAKKER H., FLORES-MOYA A., ALTA-MIRANO M., HANELT D., BISCHOF K., FIGUEROA F.L.: Impact of UVradiation on viability, photosynthetic characteristic and DNA of brown algal zoospore: implications for depth zonation. Marine Ecol.Progr.Ser. 197, 217229 (2000).