Folia Microbiol. 45 (5), 439-446 (2000) h t t p : / /www. b i o m e d , c a s . c z / m b u / f o l i a /

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

S.C. AGRAWAL, V. SINGH

Department of Botany, University of Allahabad, Allahabad. India Received 4 January 2000

ABSTRACT. The mere vegetative survival was not sufficient but suitable growth conditions were required for akinete format- ion to occur in the blue-green algae Anabeena Jyengarii, Wes- tiellopsis prolifica, Nostochopsis lobatus and in the green alga Pithophora oedogonia. In all algae, akinetes were neither for- med nor germinated in darkness, and while dim light of 300 Ix was sufficient for most of akinetes to germinate and also to maintain vegetative survival, it was not adequate for optimum akinete formation. Although akinetes of all algae could germi- nate at 35 ~ both the vegetative survival and akinete format- ion were markedly suppressed at this temperature Heat or UV

shock of any level, whether ineffective or effecting vegetative survival, did not promote akinete formation or germination in any alga tested. Akinetes of all algae under study were relati- vely tolerant to heat and also to some extent to UV. Both wet and dried akinetes of all algae were equally UV tolerant. In all algae, the viability of both wet and dried akinetes decreased more or less equally with storage time, but the decrease was more drastic when storage temperature was progressively low- ered from 20 to 0 ~ Hence the akinetes can tolerate dryness but not frost.

Earlier studies indicated that akinete formation in some algae was not favored but inhibited by desiccation (Agrawal and Singh 1999a, b). Since in nature desiccation of algae cells is usually associated with exposure of the cells to high light intensities and high temperatures, it is important to know how such exposures influence akinete formation. The impact of darkness, low light intensities, high temperatures associated with high light intensities, heat shock and UV exposure on vegetative survival, akinete formation and germination has been demonstrated in three blue-green algae Anabcena iyengarii, Westiellopsis prolifica and Nostochopsis lobatus, and in one green alga Pithophora oedogonia. Other features under investigation have included viabili ty of wet and dried akinetes stored for different time periods at different conditions, and their UV tolerance.

M A T E R I A L S AND M E T H O D S

The algae, the mode of their akineteformation and germination, the culture methods and the culture conditions were the same as described by Agrawal and Singh (1999a,b). Liquid cultures o f all algae adjusted at pH 7.5 were maintained under controlled culture conditions in a culture chamber at 22 + 1 ~ and light intensity of ca. 2 klx from daylight fluorescent tubes for 16 h per d. Maximally about 10 % vegetative cells in A. iyengarii, 99 % in W. prolifica, 97 % in N. lobatus and 99 % in P. oedogonia differentiated into aki- netes within 60 d o f inoculation. During akinete differentiation the entire vegetative cell was converted into an akinete in blue-green algae while in P. oedogonia a part o f the cell was converted into an akinete and the rest remained vegetative. When transferred to fresh culture media, most o f the freshly formed akinetes harvested from old culture media, germinated progressively within 25-30 d to form new trichomes or fila- ments (Fig. 1 A-D) .

Vegetative survival, akinete formation and germination at different light intensities and tempera- tures. Actively growing vegetative trichomes - vegetative filaments - o f all blue-green algae and the young vegetative filaments o fP . oedogonia, as well as their freshly formed akinetes were inoculated separately into culture tubes containing liquid media and exposed to different light intensities and temperatures by adjusting the distance o f the culture tubes from 100 W Philips incandescent bulb acting as a continuous light and heat source for 1 d. The different light intensities were measured with the help of a Lutron lux-meter. To obtain zero lux-light intensity, inoculated culture tubes were kept in complete darkness at room temperature. Some o f the inoculated culture tubes kept in controled culture chamber at 22 + 1 ~ and 2 klx light intensity for 16 h per d served as controls.

The percentage o f vegetative survival in P. oedogonia was determined by counting the number of surviving vegetative cells out of the total number o f vegetative cells, in the other algae by counting the hum-

440 sc . AGRAWAL and V, SINGH Vol. 45

ber of surviving vegetative filaments out of the total number of filaments. In all algae, percentage akinete formation was estimated by counting the number of akinetes formed out of the total number of vegetative cells, and the percentage germination of inoculated akinetes by counting the number of akinetes showing emergence of protuberances out of the total number of akinetes inoculated.

D

Fig. 1. Akinete germination in A. tyengarii (A), W. prolifica (B), A( lobatus (C) and P. oedogonia (D); magnification x 165.

2000 AKINETES OF ALGAE AND EFFECT OF LIGHT, TEMPERATURE, AND HEAT SHOCKS 441

Effect of heat shock on vegetative survival, akinete formation and germination. Actively growing vegetative filaments and freshly formed akinetes of all algae suspended separately in sterilized distilled water adjusted to pH 7.5 were kept at different temperatures at 40-50 ~ aliquots withdrawn at different time intervals (5~50 rain or more) were inoculated into culture media, placed in a culture chamber under normal culture conditions and scored as usual at intervals for percentage of vegetative survival, akinete formation and akinete germination. Vegetative filaments and akinetes not subjected to any heat shock and placed similarly in culture chamber served as controls.

Effect of UV treatment on vegetative survival, akinete formation and germination, and on viability of wet and dried stored akinetes. Actively growing vegetative filaments and freshly formed akinetes of all algae placed separately in I0 mL sterilized distilled water spread over Petri dishes (diameter 90 ram) were exposed to UV light from a Philips germicidal UV lamp with main output at 254 nm and a fluence rate of 3.2 W/m 2. On increasing the time of exposure (5-40 min), the energy fluence of UV light increased from 0.96 to 7.68 kJ/m 2. The irradiated materials were separately centrifuged, transferred to fresh culture media, kept in darkness for 1 d to avoid photoreactivation and then maintained in a culture chamber under normal light-dark cycle. They were examined to determine the percentage of vegetative survival, akinete formation and germination. Similarly treated unirradiated materials served as controls.

To determine whether wet and dried stored akinetes differed in UV tolerance, wet akinetes (kept in sterilized distilled water) and dried akinetes (blot-dried and kept over fused CaCI2 in desiccators) of all algae stored for six months at 20 ~ in darkness were similarly irradiated with UV light (5 and 10 min), then inoc- ulated into fresh culture media, kept in dark for 1 d and place in the culture chamber to score periodically the percentage of germination. Similarly stored and treated unirradiated akinetes served as controls.

Viability of wet and dried akinetes stored for different time periods under different conditions. The viability of akinetes of all algae either suspended in sterilized distilled water (wet akinetes) or blot-dried, placed on a filter paper and kept in desiccators over fused CaCI 2 (dried akinetes) at 20 ~ in the light (10 h duration at ca. 1.4 klx intensity), or 20, 12 and 0 ~ in the dark, and stored for various time periods of 1 to 6 months was determined by estimating the percentage of germinated akinetes 15 d after inoculation in liquid media under controlled culture conditions.

RESULTS AND DISCUSSION

Vegetative survival, akinete formation and germination at different light intensities and temperatu- res. Vegetative filaments of W. prolifica, N. lobatus and P. oedogonia survived darkness as well as dim light of 100 lx better than those ofA. iyengarii, but in no alga were akinetes formed in total darkness. A minimum of 300 lx light intensity at 29 ~ initiated the process to some extent in W. prolifica while 100 lx sufficed (to a very reduced extent) in N. lobatus and P. oedogonia (Table I). A. iyengarii vegetative filaments, although alive at 300 Ix and 29 ~ did not form any akinetes even after 45 d of incubation. Thus low light intensity, although sustaining vegetative survival, did not favor akinete formation in any of the algae tested. However, a reduction in the availability of light or transfer of the culture from light to darkness have been shown to stimulate akinete formation (under laboratory conditions) in Nostoc sp. and Anabaena cylindrica (Sutherland et al. 1979; Nichols et al. 1980).

Although Anabaenaflos-aquae grew even at 39 ~ (Novak and Brune 1985), and A. doliolum showed higher growth rate and sporulation at 32 ~ than at 25 ~ (Pandey and Kashyap 1987), we demonstrated that all vegetative filaments ofA. iyengarii died without any akinete formation after 15 d of incubation at 500 Ix light and 31 ~ while those of W. prolifica and N. lobatus died after 30 d and P. oedogonia cells died after 15 d of incubation after producing about up to 50 % akinetes at 2 klx and 35 ~ relative to controls (Table I). Thus along with vegetative survival, akinete formation also decreased at high temperature in all the algae.

Light has been found to be required for akinete germination in A. cylindrica (Fay 1969), A. varia- bilis (Braune 1979), Stigeoclonium pascheri (Agrawal 1984), Nodularia spumigena (Huber 1985) and A. circinalis (Van Dok and Hart 1997). In our study no alga showed akinete germination in darkness, and a minimum of 300 lx light at 29 ~ was required to initiate the process comparable to controls. In all algae, the vegetative survival and akinete formation, but not akinete germination, were inhibited or totally suppressed at 2 klx light intensity and 35 ~ (compared to controls). Akinete formation is therefore more sensitive to high temperatures than akinete germination. All algae died without any vegetative survival, akinete formation and germination at 3 klx and 41 ~ Since akinetes are not formed at high temperature, the akinete forming blue-green algae are generally absent from the hot spring flora (Anagnostidis 1961).

442 S.C AGRAWAL and V. SINGH Vol. 45

Table 1. Percentage of vegetative survival (S), akinete formation (F) and germination of akinetes (G) at different light intensi- ties (0-3 klx) and temperatures in A. iyengarii, ~ prolifica, N. lobatus and P. oedogonia a

S, F, G Days after Controlb inoculation

Cultures maintained at

0 0.1 0.3 0.5 2 3 29oc 29~ 29~ 31•176 35•176 41•176

A. iyengarii

S 15 100 10 50 100 50 0 0 30 - 0 30 100 0 - - 45 100 10 80 - - -

F 45 5 0 0 0 0 0 0 G 13 21 0 0 38 26 20 c 0

23 65 0 0 48 50 c 41 0

W. prolifica

S 30 100 100 d 100 100 99 40 0 45 100 100 70 100 50 0 -

F 45 72 0 0 13 28 30 0 G 13 22 0 0 30 14 28 0

23 76 0 1 50 30 60 0

N. l oba tus

S 30 100 100 d 100 100 100 58 0 45 100 100 75 100 70 0 -

F 45 61 0 5 10 31 29 0 G 13 15 0 0 25 28 38 0

23 52 0 1 60 33 66 0

P. o e d o g o n i a

S 15 99 100 100 100 58 40 0 45 98 0 100 100 8 0 -

F 15 71 0 0 0 7 10 0 45 82 0 7 25 16 30 -

G 13 28 0 0 30 30 33 0 23 40 0 8 39 31 40 0

aRounded means of three replicates. CAll germlings died. bMaintained at 22 • 1 ~ and 2 klx light for 16 h per day. dFilaments became thin and faint.

Effect of heat shock on vegetative survival, akinete formation and germination. Heat shock (42 ~

for 1 h or 45 ~ for �89 h) proved lethal to vegetat ive survival , akinete formation and germinat ion in A. iyen- garii. It was most ly ineffect ive or less effect ive in W. prolifica, N. lobatus and P. oedogonia and only expo-

sure to 48 ~ for �89 h had any effect. In P. oedogonia, although all vegetat ive cel ls died without any akinete

formation, the akinetes were able to germinate when exposed to 45 ~ for 1 � 8 9 h (Table 1I). The heat sen-

sitivity sequence therefore was A. iyengarii > P. oedogonia > W. prolifica, N. lobatus. Exposure tO 50 ~ for

5 min proved lethal to vegeta t ive cells and akinetes o f all algae. In almost all algae, heat shocks which did

not affect vegeta t ive survival , also did not affect akinete formation or germination. Inhibitory shocks were

slightly less deleterious for akinete germinat ion than for vegeta t ive survival. Hence , the akinetes were slight-

ly more heat resistant than vegeta t ive cells. The same behavior was found in the green alga Stigeoclonium pascheri (Agrawal 1985). On the other hand, Glade (1914) found that akinetes o f Cylindrospermum licheni- forme were much more heat tolerant than vegetat ive cells.

Effect of UV treatment on vegetative survival, akinete formation and germination and on viability of wet and dried stored akinetes. U V light inhibited the formation o f gonidia in Volvox carteri (Kochert and

Yates 1970) and reproduct ion in Oedogonium cardiacum (Parker and Horsley 1972), and decreased akinete

germinat ion in Nodularia spumigena (Pandey 1984). We demonstrated that increasing U V flux rates progres-

s ively decreased or comple te ly inhibited vegetat ive survival , akinete formation and germinat ion in all algae,

A. iyengarii being the most U V sensitive. In all algae, akinetes were compara t ive ly more U V tolerant than

vegeta t ive cells (Table III), but both wet and dried akinetes were equally U V tolerant (Table IV). In blue-

2000 AKINETES OF ALGAE AND EFFECT OF LIGHT, TEMPERATURE, AND HEAT SHOCKS 4 4 3

green algae, the p r e s e n c e o f the ext race l lu lar p i g m e n t s cy tonemin (Garc i a -P iche l et al. 1992) and myco-

spor ine - l ike c o m p o u n d s ( G a r c i a - P i che l and C a s t e n h o l z 1993) were f o u n d to be i m p o r t a n t for s c r e e n i n g

U V light.

T a b l e I1. Effect of heat shock (42-48 ~ duration 15-120 min) on per cent survival (S), akinete formation (F), and ger- mination of akinetes (G) in A. iyengarii, W prolifica. N. lobatus and P. oedogoma a

42 45 48 S, F, G Days after Control

inoculation 15 30 60 15 30 90 120 15 30

A. iyengarii

S 15 100 100 10 0 10 0 - 1 0 F 30 1 2 0 - 0 - - - 0 -

45 4 4 1 - 1 - - - 0 -

G 13 45 40 35 0 20 0 - - 10 0 23 70 59 41 0 30 0 - - 10 0

W. prolifica

S 15 100 - - 100 - 100 45 43 90 0 F 30 38 - - 31 - 29 20 22 30 -

45 60 - - 50 - 60 41 39 60 - G 13 48 40 28 40 41 30 10 17 29 0

23 75 70 66 55 59 40 24 26 48 1

N. loba tus

S 15 100 - - 100 - 100 30 25 95 0 F 30 10 - - 14 - 15 6 4 11 -

45 48 - - 46 - 40 23 20 50 - G 13 20 25 20 26 20 28 20 15 25 0

23 69 58 45 48 56 38 29 24 45 0

P. oedogonia

S 15 t00 - I00 50 100 80 0 0 10 0 F 30 79 - 60 25 15 10 - - 5 -

45 80 - 80 65 48 50 - - 5 - G 13 25 23 20 18 28 25 15 11 28 0

23 45 35 38 30 40 40 30 22 35 0

aRounded means of three replicates.

Viability o f wet and dried akinetes stored for different time periods under different conditions. I r respect ive o f s torage condi t ions , the pe rcen tage o f ge rmina t ion o f bo th wet and dr ied akine tes o f all algae

decreased more or less equal ly wi th an increase in s torage t ime, bu t more rapid ly w h e n s torage t empera tu re

was lowered ( f rom 20 to 12 and 0 ~ In this r ega rd P. oedogonia ak ine tes were more sens i t ive t h a n those

o f any o f b lue -g reen a lgae used. Hence the ak ine tes o f all algae were able to su rv ive d ryness bu t no t the frost

wh ich is assoc ia ted wi th phys ica l abs t rac t ion o f water f rom the p ro top lasm. U n d e r na tura l condi t ions ,

P. oedogonia o v e r w i n t e r s as ak ine tes and v e g e t a t i v e f i l amen t s and is still p h o t o s y n t h e t i c a l l y c o m p e t e n t

(Spencer et al. 1981). Schere r and Potts (1989) desc r ibed the synthes is o f un ique pro te ins by f ie ld-col lec ted

Nostoc commune upon repea ted des icca t ion and p re sumed that they inc reased the des icca t ion to le rance .

The abi l i ty o f ak ine tes to pe r enna t e for m a n y years and subsequen t l y ge rmina te ( Y a m a m o t o 1975;

L iv ings tone and Jaworsk i 1980; Sili et al. 1994) let to the b e l i e f tha t they were impor tan t in the life cycle o f

Nostocales as m a n y species ove rwin t e r as vege ta t ive f i l aments (Reyno lds 1972; Kappers 1976; Ba rb i e ro and

Welch 1992). However , vege ta t ive f i l aments o f all a lgae used in our s tudies were m u c h more f ros t sens i t ive

than akinetes (Agrawa i and S ingh 1999a,b) . It was poss ib le tha t the p roper t i es o f m u d d y or c lay bo t tom

sed iments exposed to f reez ing condi t ions m ay reduce the adverse ef fec ts o f frost on ak ine tes or vege ta t ive

cells bur ied wi th in t hem.

444 S.C. AGRAWAL and V. SINGH Vol. 45

Table i l l Effects of UV treatment (5-40 min) on per cent vegetative survival (S), akinete formation (F) and akinete germination (G) in A. iyengar#, I4( profilica, N. lobatus and P. oedogonia a

Days a~er S, F, G inoculation Control 5 10 20 30 40

A. i y e n g a r i i

S 15 100 71 8 0 0 0 F 45 2 2 0 - - - G 13 50 28 0 0 0 0

23 70 38 16 3 0 0

W. p r o l i f i c a

S 15 100 100 20 0 0 0 F 45 49 45 8 - - - G 13 48 30 10 0 0 0

23 75 43 30 6 5 1

N. l o b a t u s

S 15 100 100 18 0 0 0 F 45 40 50 8 - - - G 13 20 22 20 2 0 0

23 68 60 48 8 5 0

P, o e d o g o n i a

S 15 100 100 10 0 0 0 F 45 70 51 6 - - - G 13 25 26 10 5 0 0

23 48 35 24 15 7 5

aRounded means ofthree ~plic~es.

Table IV. Effect of UV treatment (5 and 10 min) on per cent germination of wet (W) and dried (D) akinetes ofA. iyengarii, V~ prolifica, N. lobatus and P. oedogonia stored for six months at 20 ~ in darkness a,b

Days after Alga inoculation

Control 5 10

W D W D W D

A. iyengarii 13 20 25 20 13 1 1 23 48 58 41 40 10 12

W prolifica 13 13 28 20 20 1 5 23 40 51 41 50 21 31

N. lobams 13 20 30 15 15 5 10 23 41 49 38 40 15 22

~oedogon~ 13 20 25 15 21 2 5 23 49 50 35 40 18 20

awet akinetes were stored in sterilized distilled water, dried akinetes over fused CaCI 2. bRounded means of three replicates.

2000 AKINETES OF ALGAE AND EFFECT OF LIGHT, TEMPERATURE, AND HEAT SHOCKS 445

Table V. Per cent germination of dried akinetes ofA. tyengarii, W prolifica, N. lobatus and P. oedogonla stored for 1-6 months in desiccators over fused CaCI 2 at different light conditions and temperatures (20, 12 and 0 ~ a

Storage conditions

Storage dark time, month light b

20 12 0

A. i y e n g a r i i

1 50 33

2 52 50

3 52 50

5 35 40

6 21 25

51 49 14 50 48 13

50 49 0 58 48 13

46 36 0 48 38 3

38 25 0 40 30 0

20 20 0 25 15 0

W. p r o l i f i c a

1 48 68 46 12 48 50 47 20

2 47 47 47 2 48 48 49 8

3 39 40 38 1 44 47 39 0

5 30 39 18 0 34 47 20 0

6 28 40 10 0 41 48 19 0

Storage conditions

Storage dark time, month light b

20 12 0

N. l o b a t u s

1 47 49 41 10 48 48 49 19

2 43 48 41 10 45 50 45 19

3 40 48 40 8 46 50 46 7

5 38 30 17 0 45 46 15 2

6 22 28 l0 1 42 44 18 2

P. o e d o g o n i a

1 55 70 1 0 50 68 0 0

2 56 71 1 0 58 75 0 0

3 45 30 0 0 40 40 0 0

5 40 30 0 0 42 36 0 0

6 29 30 0 0 38 35 0 0

aMeasurement 15 d after inoculation in liquid media; controls were maintained in sterilized distilled water and kept under storage conditions; all values represent rounded means of three replicates;first lines - control, second lines - dried stage.

bStorage under 10-h illumination with fluorescent light of 1.4 klx at 20 ~

similar

The authors thank the Head, Department of Botany, University of Allahabad (India) for providing laboratory facilities and the University Grants Commission. New Delhi, for financial assistance in the form of a research project.

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