Zoosporangia Survival, Dehiscence and Zoospore

Folia Microbiol. 49 (5), 549–556 (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 Bold’s basal medium at pH 7.5, temperature of about 22 °C and light intensity of about 40 μmol m–2 s–1 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 0.2–0.4 mol/L NaCl, 5–60 min blot-dryness of fila-ments), 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 (0.96–3.84 kJ/m2), lack of all nutrients from liquid medium (double distilled water), darkness, and presence of “heavy” metals (1–25 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 pro-cesses. 3-IAA at 50 and 400 ppm induced zoosporangial papilla to grow into a tube-like projection of about 30–120 μm 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 envi-ronmental 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 O’Kelley 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 perfor-mance 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 Häder 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 survi-val, zoospore formation, zoosporangia dehiscence, zoospore speed and time period of motility in Rhizoclo-nium hieroglyphicum (C. AGARDH) KÜTZING.

MATERIAL AND METHODS

Algal material and growth conditions. R. hieroglyphicum was collected while growing firmly atta-ched to cement walls in a running freshwater shallow tank on the university campus (University of Allaha-bad, India). Clonal cultures were raised through germinating zoospores and were maintained under controled culture conditions in Bold’s 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 μmol m–2 s–1 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 15–20-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 zoo-sporangium 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 μm (Fig. 1G). Most of the zoospores of a zoosporangium escape into the medium, continuously or intermittently, within 3–4 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 2–3 d and thereafter disintegrated. The released zoospores swam in the me-dium for 30–120 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 spread-ing it on solid BM containing 2 or 4 % agar, or inoculating it in liquid BM with 0.2–0.4 mol/L NaCl. Another part of the algal material was completely blot-dried for 5–60 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 5–10.5 (determined with Sys-tronic digital pH meter) prior to autoclaving by adding 1 mol/L HCl or 2 % NaOH and then kept in the cul-ture chamber.

Temperature shock. Algal material suspended in BM was placed at 5–50 °C; aliquots were with-drawn after 5–60 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 1–25 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 100–2000 ppm. 3-IAA (100 %; Loba-Chemie, India) was dissolved separately in a minimal volume of 80 % ethanol (2–3 drops) and mixed slowly into a known amount of BM to prepare the hormone solution of desi-red concentrations of 50–400 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 brown-ish in color) out of about 8000–9000 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 800–980 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.


Zoospore speed was determined by following their path of movement (which was very irregular) with res-pect to microscale and was expressed in μm/min. About 1000 zoospores were assessed for each of three repli-cates in each case. In control algal material the zoospore speed range was between 120 and 200 μm/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 pri-mordia, 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 Tetraed-ron sp. (Starr 1954a,b). Water stress inhibited zoosporangia formation and zoospore germination in Clado-phora 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, oogo-nium 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 sub-stances 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 PZNb RZR ZS ZMR0

C o n t r o l c

Liquid BM 100 22 800–950 35–45 120–200 30–150

W a t e r s t r e s s

Agarized BM, % 2 92 0 ZF 0 – –0 4 90 0 ZF 0 – –0

NaCl in liquid BM, mol/L

0.2 88 12 800–900 40–45 120–200 30–900 0.3 79 2 450–500 05–10 080–140 100 0.4 70 0 ZF 0 – –0

Blot-dryness of filaments, min

5d 30 2.5 800–900 15–20 096–160 100 10e 20 1.5 600–800 05–70 054–900 05–100 20 19 0 0 – – –0 60 13.5 0 0 – – –0

p H o f l i q u i d B M

5.0 45 0 0 – – –0 6.5e 62 7.5 800–900 20–22 120–200 10–130 7.5 100 24 800–920 40–45 120–200 30–150 9.5 92.5 6 800–900 20–25 066–110 05–100

10.5e 75 1 800–900 03–10 084–120 05–100

T e m p e r a t u r e s h o c k i n l i q u i d B M

Temperature, °C min 5 5e 96 0.5 500–600 20 108–180 30–450 5 10e 85 0 ZF 0 – –0 5 30 51 0 0 – – –0


15 60 100 21 800–950 32–40 120–200 30–900 35 5 73 0 ZF 0 – –0 35 60 54 0 ZF 0 – –0 40 5 25 0 0 – – –0 50 5 1 0 0 – – –0

U V e x p o s u r e

Dose, kJ/m2 0.96d 96 2 800–950 35–45 120–200 30–900 1.92 85 0 ZF 0 – –0 3.84 50 0 0 – – –0

L a c k o f a l l n u t r i e n t s

Double-distilled water 10 0 0 – – –0

D a r k n e s s

Zero light intensity 97 0 ZF 0 – –0

M e t a l s i n l i q u i d B M , p p m

Copper 1e 96 2.5 300–450 10–15 042–700 05–100 5 90 0 ZF 0 – –0

25 73 0 ZF 0 – –0

Iron 1e 98 1 250–400 05–10 036–600 50 5 90 0 ZF 0 – –0

25 78 0 ZF 0 – –0

Zinc 1e 92 1.5 300–400 8–10 040–500 50 5 80 0 ZF 0 – –0

25 67 0 ZF 0 – –0

Mercury 1 35 0 0 – – –0 5 6.5 0 0 – – –0

25 0 – – – – –0

Nickel 1 92 8 600–750 10–20 120–160 300 5 77.5 3.5 400–550 05–10 084–140 100

25 65 0 ZF 0 – –0

Cobalt 1e 92 1 250–300 05–10 030–450 50 5 83 0 ZF 0 – –0

25 74 0 0 – – –0

O r g a n i c s u b s t a n c e s i n l i q u i d B M , p p m

Captan 200 82.5 1 400–500 05–80 080–130 50 400 61 0 0 – – –0 600 50 0 0 – – –0

DDT 200 45 0 0 – – –0 400 30 0 0 – – –0 600 16 0 0 – – –0

continued


2,4-D 200 100 10 950–980 35–45 120–200 30–120 800d 90 2.5 400–550 25 108–180 10–200

1000 84 0 ZF 0 – –0

3–IAA 0 100 20 900–950 37–45 120–200 30–900

50 100 0f ZF 0 – –0 400 100 0f ZF 0 – –0

Urea 200g 100 50 950–980 50–60 120–200 90–150 400g 100 36 800–860 30–35 120–200 15–300

1000 100 10 450–550 25–32 108–180 15–200 2000e 100 2 250–430 10 060–100 5

Thiourea 100 14 0 0 – – –0 200 5 0 0 – – –0

aZOS (in %), ZOD (% with respect to ZOS), RZR (% of zoospores released with respect to total present per min), ZS (in μm/min), ZMR (in min); all readings on 10 d after inoculation except otherwise indicated; rounded mean of 3 replicates; for details see Mate- rial 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 μmol m–2 s–1 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 μm 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 de-hiscence. 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 adver-sely 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 6.4–9.5 was most favorable to the mo-vement 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 7–8 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 20–25 °C. The alga exists only in the vegetative stage and did not show any zoosporangial stage during July–August when the water temperature was 30–31 °C. However, no zoosporangia present in the alga dehisced during mid-De-cember to January when the water temperature was 6–14 °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 O’Kelley 1966), in Ulothrix zonata near 20 °C (Graham et al. 1985) and in Coleochaete scutata at 20–25 °C (Graham et al. 1986). Zoosporoge-nesis in green algae C. glomerata (Hoffmann and Graham 1984) and C. scutata (Graham et al. 1986) de-clined 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 (Häder and Häder 1988), survival rate in some brown algal zoospores (Wiencke et al. 2000; Swanson and


Druehl 2000) and cell division in Chroococcus sp., Gloeocapsa sp. and Aphanothece sp., formation of hetero-cysts 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 (O’Kelley and Deason 1962), Botrydiopsis sp., Bumilleriopsis sp. and Pseudollumilleriopsis sp. (Pecora and Russell 1973) and Chlorococcum echinozygotum (O’Kelley 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 zoo-spores 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 1–25 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 expo-sure 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 200–600 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 800–1000 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 μm 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 zoo-sporangia 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. hiero-glyphicum 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 μmol m–2 s–1 for 16 h a day. Any alteration in the physical and chemical envi-ronment suppressed them variously. It is also evident that zoosporangia dehiscence rather than zoospore for-mation 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 me-dium.

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Zoosporangia Survival, Dehiscence and Zoospore

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