Life Cycle of Peridinium Sp. B3 (Dinophyceae) Isolated from Lake Begnas, Nepal

Nihon Biseibutsu Seitai Gakkaiho Vol. 1, No. 1, 19-27, 1986

Life Cycle of Peridinium sp. B3 (Dinophyceae) Isolated from Lake Begnas, Nepal

YOSHIHIKO SAKO1, MASAMI NAKANISHI2, TOSHIFUMI KONDA3, YUZABURO ISHIDA1 HAJIME KADOTA4, KESHAB SHRESTHA5, HEMANTA R. BHANDARY5

and RAJENDRA L. SHRESTHA5

1. Laboratory of Microbiology, Department of Fisheries, Faculty of Agriculture, Kyoto Uriiversity, Kyoto 606, Japan. 2. Otsu Hydrobiological Station, Faculty of Science, Kyoto Un iversity, Otsu. lapan. 3. Department of Bacteriology, National Institute of Health, Tokyo, Japan. 4. Department of Fisheries, Faculty of Agriculture, Kinki University Osaka, Japan. 5. Natural History Museum, Tribhuban Umversity, Nepal.

Abstract: Encystment and excystment in the life cycle of a dinoflagellate Peridinium sp. B3, which was isolated from Lake Begnas in the Pokhara Valley, Nepal, were investigated by use of a clonal population. The maximum cell concentration was 3103 vegetative cellsml-1 in BL medium after about 4 weeks at 30C. Sexual reproduction of the cells was induced after the cells in late logarithmic phase were inoculated into nitrogen and phosphorous deficient medium, and also was spontaneously induced in BL medium. Sexual reproduction was isogamous and homothallic. The cycle was initiated by gamete formation, rn which gametes were produced by binary asexual divisions of the vegetative cells. Fusing pairs were formed by the mid-ventral union of gametes. The planozygote retained two red bodies and remained motile for about 2 weeks. Two weeks after the planozygote lost motility, the zygotes enlarged and became dark in color. The theca of the planozygote split around the girdle region and the hypnozygote (cyst) was produced. The matured cysts were dormant and remained viable for at least 1 month at 10C in darkness following maturation for 3 months at 30C. The dark-cold treated cysts excysted synchronously when the incubation temperature was raised to 30C. The protoplast emerged from the cyst wall by amoeboid movement. The excysted cell had two red bodies. This cell divided within 24-48h into 2 daughter cells each with a red body.

Key words: Peridinium, cyst, life cycle, freshwater red tide

Introduction (Watanabe, 1982) and in Ananai Reservoir (Hata, 1982). For P. cunningtonii inoculated in nitro- In recent years large-scale blooms, so called gen and phosphorus-deficient medium the sexual freshwater red tides, of dinoflagellates belonging to life cycle developed in the following 5 phases: i) the genus Peridinium have often occurred in man- gamete formation; ii) fusion; iii) planozygote; made lakes (Nakamoto, 1975; Ito, 1979; Hata, iv) hypnozygote (cyst); and v) excystment (Sako 1982) and rarely in natural lakes (Home et al., et al., 1984). It was suggested that the excystment 1971; Pollingher and Serruya, 1976). For exam- of overwintering cysts of P. cunningtonii provided ples, P. cinctum in Lake Kinneret (Eren, 1969), P. a seed population for the bloom of this cunningtonii and P. penardii in Nagase Reservoir dinoflagellate in the reservoir (Sako et al., 1985). (Hata, 1982), and P. bipes in Kazaya Reservoir Although studies on sexual life cycles are very 20 SAKO et al important not only for the ecology of excystment in the life cycle of this dinoflagellate dinoflagellates but also for their taxonomy, the Peridinium sp. B3 isolated from Lake Begnas. difficulties in isolation, cultivation and mainte- nance of these algae hinder such investigations. Materials and Methods So far, sexual life cycles have been reported for only 23 of the 1,000 known species of Organism and culture dinoflagellates (Pfiester, 1984). Of these, seven A vegetative cell of Peridinium sp. B3 was iso- are members of the genus Peridinium (Pfiester, lated from Lake Begnas (ca. 650m above sea level) 1975, 1976, 1977; Pfiester and Skvarla, 1979, of the Pokhara Valley, Nepal, in September 1984 1980; Pfiester et al., 1984; Sako et al., 1984, (Fig. 1). Surface water temperature in the sampl- 1985). In the first scientific expedition "Studies ing station was 29-30C and water temperature in on Distribution, Adaptation and Evolution of the shallow inlet of the Lake was 36C. Clonal Microorganisms in Nepal Himalayas" in culture was utilized in all the experiments de- November-December 1982, three subtropical lakes scribed below. in the Pokhara Valley were surveyed to compare Cells were grown at 30C with a 14:10h light- the structure and function of phytoplankton com- dark cycle under illumination of 15,000lx. No munities in the lakes on the basis of primary bubbling or shaking of the culture was employed. production and species diversity (Nakanishi et al., Peridinium sp. B3 was grown in BL medium, the 1984). Nakanishi et al. (1984) could not detect composition of which is shown in Table 1. any species of Peridinium. In a second expedition Cell numbers were determined with a which was carried out in September-October, 1984, hemocytometer after fixation with formalin. several species of Peridinium were found only in September in Lake Phewa, Lake Begnas and Lake Observation of life cycle Rupa. In particular, in Lake Begnas two species The sexual life cycle was followed by isolating of Pridinium caused a small bloom in the middle each individual pair of fusing gametes in 0.2ml of of September. One of these species has been suc- media poured into a hole of a Nunclon Microtest cessfully cultivated, and the present study investi- Plate 96F. The basal medium with nitrogen and gates the conditions regulating encystment and phosphorus omitted was used to induce sexual

Fig. 1. Peridinium sp. B3 A, ventral view. B, Dorsal view. Life Cycle of Peridinium sp. B3 21

Table 1. Composition of BL medium for Peridinium The dark-cold treated cysts were washed twice sp. B3 with distilled water and incubated under 10,000 lx (14L: 1OD) at 30C in fresh medium. Excystment of Peridinium sp. B3 was observed with a differential interference microscope (Nikon).

Results Vegetative growth A typical growth curve of Peridinium sp. B3 in BL medium under 15,000 lx at 30C is shown in Fig. 2A. In the exponential phase the cell divided into two daughter cells. Growth rate was approx- imately 0.43 day-1 and the maximum cell concentration was 3103 vegetative cellsml-I after 4 weeks. The optimum temperature for maximum growth rate and cell yield was 30-32C in both cases (Fig. 2B).

pH 7.0 Morphological observations of encystment reproduction although some cells in the popula- Vegetative cells of Peridinium sp. B3 measured tion reproduced in the stationary phase of growth 20-24m in length and 18-22m in width, and in BL medium. The zygotes formed were checked were angular in ventral view (Fig. 3). The cell daily with an inverted microscope. The cysts had two flagella, a longitudinal one located in the formed matured at 30C after 3 months and were sulcus and a transverse one located in the cin- stored at 10C for more than 30 days in darkness. gulum. Vegetative cells surrounded by a theca

Fig. 2. Growth curve of Peridinium sp. B3 in BL medium. A, The culture was incubated at 30c with a 14: 10h light-dark cycle under illumination of 15,000lx. B, Temperature effect. All cultures were incubated at 15,000lx (14L: 10D) for 5 weeks in BL medium. 22 SAKO et al

Figs. 3-8. Light micrographs of encystment in Peridinium sp. B3. Scale bars=20m. Fig. 3. Vegetative cell. Fig. 4. Vegetative division. Figs. 5, 6. Fusion of gametes. Fig. 7. Planozygote. Fig. 8. Release of cyst (hypnozygote). Life Cycle of Peridinium sp. B3 23 had a small red body and many spines on the test tube. By excreting mucilaginous material the lower ventral side. Numerous yellow-green or cysts easily became aggregated to each other. yellow-brown chloroplasts were uniformly dis- tributed in the cytoplasm. Aggregated thecal Morphological observations of excystment debris was often observed in the cultured tubes. The matured cysts contained a large red body Vegetative cells exhibited strong positive phototax- and the yellow-green chloroplasts remained dor- is. Cell division by binary fission occurred out- mant for 3 months. The matured cysts of Peri- side the theca through the peanut-shaped stage dinium sp. B3 were stored at 30C for 3 months and shown in Fig. 4. The dividing cell showed no then maintained for 1 month at 10C in darkness. motility. The cysts which experienced such a treatment Sexual reproduction was induced when the cells retained a large red body and various granules. in late logarithmic phase were inoculated into N- The dark-cold treated cysts synchronously excyt- and P-free medium, and also spontaneously occur- ed about 7 days after when the incubation tempera- red in the stationary phase in BL medium. ture was raised to 30C. The cysts without previ- Gametes were formed within a few days after ous cold treatment scarcely excysted under any inoculation into N- and P-free medium by binary other sort of conditions. Excystment was obser- fission. In this species the gametes appear mor- ved with a differential interference microscope phologically identical to the parent vegetative cell (Figs. 10-13). The initial phase of excystment except that they were usually smaller. Gametes involved breaching of the cyst wall, while in the ranged from 15 to 18m in length. Gametes were second phase the protoplast was extruded through lighter in color than vegetative cells, and possessed the resultant aperture. A dum-bell shaped proto- thin thecae. Gametes were isogamous (Figs. 5 plast escaped from the cyst wall. After 1 to 2min and 6). Many fusing gametes were observed dur- from breaching of the cyst wall half of the proto- ing the dark cycle. Fusing pairs formed by the plast was outside the cyst wall (Fig. 10). Both the mid-ventral union of gametes were observed. interior and exterior portion included a large red Sexual fusion required about 2h from gametic body (Fig. 11). The greater part of the protoplast contact to completion. Sexual reproduction was emerged from the cyst wall by amoeboid move- thus isogamous and homothallic. ment after 3min, its shape became slender and Within 30h the planozygotes readily developed long (Fig. 12), and shortly thereafter it became its own thecae (Fig. 7). The planozygotes had entirely free (Fig. 13). This process was facilitat- two longitudinal flagella and retained two red ed by a colorless mucilage which surrounded the bodies. The planozygotes could remain motile protoplast within the cyst wall before excystment. for about 2 weeks after fusion. During that time The excysted cell was enveloped by the mucilage, the planozygotes slowly enlarged to 23-27m and but after a few minutes the cell freed itself and became dark in color. Two weeks after the plano- swam away. Longitudinal and transverse flagella zygotes lost motility, they settled on the bottom of were observed at this stage of excystment (Figs. 11, culture vessel and shed their thecae (Fig. 8). 12, and 13). The initial free-swimming cell was After the thecae split at the girdle region, the cysts rocket-shaped and lacked a clear transverse fur- (hypnozygotes) were released. The cyst of Peri- row. The excysted cell had two red bodies, and dinium sp. B3 which was about 18m in diameter, divided within 24-48h into 2 daughter cells (ordi- was round and the surface of the cyst was relatively nary vegetative cells) each with a prominent red smooth (Fig. 9). The cyst contained yellow- body (Fig. 14). brown or yellow-green chloroplasts and retained a The above described life cycle of Peridinium sp. red body. Cysts always sank to the bottom of the B3 is schematically illustrated in Fig. 15. 24 SAKO et al

Figs. 9-13. Series of micrographs showing short-time excystment process of Peridinium sp. B3. Cyst which had been stored at 10C for 30 days in the dark excysted after the incubation temperature was raised to 30C, at 15, 000 Ix in BL medium. Scale bars=20Pm. Fig. 9. Cyst. Fig. 10. Amoeboid excysting cyst enveloped by a colorless mucilage. Figs. 11, 12, 13, Excysting cyst contained two red bodies and a longitudinal and a transverse flagellum. Fig. 14. First division of an excysted cell. Scale bar=20m. Life Cycle of Peridinium sp. P3 25

ASEXUAL CYCLE SEXUAL CYCLE Fig. 15. The schematic life cycle of Peridinium sp. B3.

Discussion induced in nitrogen and phosphorus deficient medium; ii) gametes are similar in shape to vege- Between the two species (sp. B3 and sp. B4) of tative cells; iii) sexual reproduction is isogamous Peridinium which caused the small bloom in Lake and homothallic; iv) gametes fuse during the Begnas of Pokhara Valley, one (sp. B3) was suc- dark period of the LD cycle; v) planozygotes cessfully cultivated when the lake water of Lake have two large red bodies and two longitudinal Begnas was used for preparation of the BL medium flagella; vi) mature cysts are released by splitting (Table 1), but the other species (sp. B4) failed to of the girdle region of the theca of the grow in BL medium. The growth of Peridinium hypnozygote; and vii) the dark-cold treated cysts sp. B3 was very slow and the optimum temperature produce one excysted cell. The differences with for maximum cell yields was very high (30-32C), P. cunningtonii include; i) two gametes are for- in comparison with P. cunningtonii, which was med outside the parental theca of Peridinium sp. isolated from a Japanese reservoir and which has a B3 but within those of P. cunningtonii; ii) the high growth rate (0.95 day-1) and a low optimum excysted cell has two red bodies in Peridinium sp. temperature (20C) (Sako et al., 1984). Peri- B3 as opposed to one in P. cunningtonii. Sexual dinium sp. B3 was also able to grow even at 36C. reproduction of P. cinctum (Pfiester, 1975), P. This fits with observation that the surface water willei (Pfiester, 1976), and P. gatunense (Pfiester, temperature in Lake Begnas was 29-30C and 1977), which are only induced in nitrogen deficient especially the water temperature in a shallow inlet medium, is also similar to that of Peridinium sp. B3 of the Lake was 36C during the small bloom of except in regard to gamete formation and the Peridinium sp. B3. amount of red bodies, though sexual reproduction The sexual life cycle of Peridinium sp. B3 is of P. volzii (Pfiester and Skvarla, 1979) is similar to that of P, cunningtonii (Sako et al., isogamous and heterothallic with two mating 1984, 1985) in the following ways: i) sexuality is types. P. cinctum and P. willei formed a round, 26 SAKO et al dark thin-walled asexual cyst when being inoculat- Acknowledgment ed in N-free medium, but in Peridinium sp. B3 an asexual cyst was not observed. This work was supported by a Grant-in-Aid for Pfiester (1975, 1976) and Pfiester and Skvarla Overseas Scientific Survey from the Ministry of (1979) observed that the cysts of P. cinctum, P. Education, Science and Culture of Japan (Project willei, and P. volzii after remaining dormant for 2- No. 59041076 and No. 60043042). We are grate- 4 months excysted and released one excysted cell ful to Messrs. D. B. Swar and P. L. Jashi of the which retained a red body. In contrast, within 12 Fisheries Development Section of the Department h of zygote formation the cysts of P, gatunense of Agriculture, Ministry of Food, Agriculture and divided into 2 daughter cells (Pfiester, 1977). The Irrigation of Nepal, and Mr. M. Wada of the Japan cysts of Peridinium sp. B3 remained dormant for 3 Overseas Cooperative Volunteer Force for their months and then excysted producing one excysted help during the field survey. cell which retained two red bodies. In Peridinium sp. B3 we observed that the protoplast escaping References from the cyst wall by amoeboid movement was, enveloped by a colorless mucilage as in the case of Eren, J., 1969. Studies on developmentcycle of Per- P. cunningtonii (Sako et al., 1985), Woloszynskia idinium cinctum f. westii. Verh. Internat. Verein. 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Terashima, M.Watanabe and P.N. without being dependent on light and nutritional Mishra, 1984. Preliminary report on lim- nological survey in lakes of the Pokhara Valley conditions (Sako et al., 1985). von Stosch (1973) (Nepal) in November-December 1982. In, found that well synchronized excystment occurred Studies on distribution, adaptation and evolution when the cysts had been activated by cold treat- of microorganismsin Nepal Himalayas (preliminary report) (edited by H. Kadota) pp. 31-41. ment for at least 4 weeks in the dark at 6C for W. Dogura Press, Kyoto. apiculata and at 3C for G. pseudopalustre before Pfiester, L.A., 1975. Sexual reproduction of Per- they were brought back to light and higher temper- idinium cinctum f. ovoplanum(Dinophyceae). J. Phycol., 11, 259-265. atures. Such effects of cold treatment on excyst- Pfiester, A.L., 1976. Sexual reproduction of Per- ment in P. cinctum was also observed by Pfiester idinium willei (Dinophyceae). J. Phycol., 12, (1975). 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