Culture of green Noctiluca under laboratory conditions: I. Feeding behavior and sexual reproduction Thaithaworn Lirdwitayaprasit Department of Marine Science, Faculty of Science, Chulalongkorn University, Phya Thai Rd., Bangkok 10330, Thailand. Tel.(662)2185394, Fax : (662)2550780, E-mail : lthaitha@netserv.chula.ac.th Abstract Several cells of green Noctiluca were collected from Chonburi Province, the Upper Gulf of Thailand and the clonal cultures were grown in small test tubes, containing 10 ml of ESMmedium, under the light illumination of approximately 3,000 lux with D:L cycle 12:12 hrs. at 27±1 C. These cultures of Noctiluca scintillans were fed on Dunaliella salina, D. viridis, Chattonella marina and Heterosigma carterae. The symbiotic green algae, Pedinomonas noctilucae, inside N. scintillans decreased gradually for about one week after incubation and the host cell began to feed on varous kinds of phytoplankton that were used as food supply in this study. In the control culture, in which no any food organisms were supplied, the symbionts completely disappeared after incubation for about two weeks but during this period cell division in N. scintillans still occurred. Hereafter for about one month all host cells in the control culture test tube died. Sexual reproduction took place only in the cultures that fed on phytoplankton when cell density of N. scintillans reached about 10 cells/ml. After loosing the symbionts, green Noctiluca in tropical area showed feeding behavior and sexual reproduction process in the similar manner as reported in Noctiluca that found in temperate area. Introduction Noctiluca scintillans and Trichodesmium erythraeum are the main causative red tide organisms in the Gulf of Thailand (Charernphol, 1958; Suvapepun, 1989 and 1992). Oxygen depletion and high ammonia concentration in the water during the blooms of Noctiluca sometimes caused damage impacts on shrimp farms in the coastal area(suvapepun,1989). Okaichi and Nishio (1976) showed that the high cellular content of ammonia in Noctiluca was toxic to fish. Although the feeding habit and sexual process in this species have been studied well by a number of authors (Enomoto, 1956; Zingmark, 1970; Takayama, 1977; etc.) but those studies were carried on the Noctiluca without the symbiont. The information on the green Noctiluca was quite rare (Sweeney, 1971) which was due to the difficulty to culture. The purpose of this study is to provide some basic biological informations of this green Noctiluca. Materials and Methods Noctiluca have been collected from Chonburi Province, the Upper Gulf of Thailand, and concentrated by plankton net of 125 µm mesh size. Cells were isolated in and washed through a sery of autoclaved filtered seawater. Only a single cell was placed in each culture
test tube containing of 10 ml of ESM-culture medium which was prepared by the method described by Okaichi et al.(1983). All culture tubes were kept in the incubator under the light illumination of approximately 3,000 lux with D:L cycle 12:12 hrs at 27±1 o C. These cultures were fed on various kinds of food organisms which were Dunaliella viridis, D. salina, Chattonella marina and Heterosigma carterae. Growth rates of Noctiluca have not been measured in this study. Some Noctiluca cells from the culture test tube were placed into multi-well plates for observation of feeding behavior and some cells that undergoing gametogenesis were also isolated and placed in other multi-well plates for observation of sexual reproduction under inverted microscope. Results Feeding behavior During the culture period,in all test tubes the green symbiotic algae (Pedinomas noctilucae) inside Noctiluca cells decreased gradually and finally disappeared. The colour of this dinoflagellate changed from green to pale-pink with the food particles have in its food vacuole (Fig. 1). Noctiluca could feed on all kinds of phytoplankton which were used as the food supply for this dinoflagellate. Dunaliella viridis seemed to support the highest cell density of Noctiluca, although any growth rates were not measured in this study. This small food and other phytoplankton used in this study were stuck to the motile tentacle and then brought to the mouth groove. Phagocytosis, the enclosure of a food particle in a membrane-covered vacuole in which digestion takes place, occur red at the mouth (Fig. 2). Noctiluca fed on other Noctiluca cells and some suspended solid in the culture were also observed. The predator cell came close to the prey cell and turned its position for the mouth groove to attach the target cell and then engulf the prey cell (Fig. 3). Other suspended materials in the culture medium were also ingested by Noctiluca as the same process as described above (Fig. 4). Sexual Reproduction In Noctiluca cultures at cell concentration of about 10 cells/ml, some cells undergoing gametogenesis were observed. The cell morphology changed to more spherical shape, mouth and tentacle deduced and finally disappeared. This cell called gametocyte mother cell. The nuclear mass was observed at the cell surface of the mother cell (Fig. 5.1). A few hours later, nuclear mass divided to two (Fig. 5.2), then nuclei cells divided more and were arranged in a distinct tetrad (Fig. 5.3). Hereafter several further divisions of the tetrad nuclei took place and might end at 128 nucleate stages with less or more number of nuclei were sometimes observed, probably due to the size limitation of the gametocyte mother cell. At this stage, the gamete bodies were observed on the surface of the mother cell and the flagella were moving (Fig. 5.8). The gamate was ovoid and flattened with diameter of about 10µm. These gamates were released from the mother cell and swam in the medium culture. Once, the two isogamate had conjugated in the culture medium, the conjugated gametes sunk to the bottom of culture vessel. A few hours later, the round shape zygote without flagella was formed (Fig. 6.1). After a few days, this zygote has developed to the new Noctiluca cell as shown in Fig. 6.2-6.3.
Discussion and Conclusions The gradual decrease of Pedinomonas noctilucae in Noctiluca cell was probably due to unfavorable conditions for the growth of this symbiotic green algae and/or ingestion by the host. In natural samples, very few food particles were observed in food vacuoles of green Noctiluca. This evidence suggested that in natural condition green Noctiluca need not any food supply from surrounding seawater. Probably, some dissolved organic compounds released from the symbiont and/or ingestion of the symbiont itself were enough for growth of the host. In laboratory culture, the phagocyt ic process in this green Noctiluca was observed during the period of decreasing of P. noctilucae. It was probably due to the low food supply from the symbiotic green algae. The feeding behavior of Noctiluca on various kinds of foods were especially well studied (Enomoto, 1956; Sweeney, 1971; Takayama, 1977; Kimor, 1979; etc.). In this study, Noctiluca could feed on non-living materials, among others and various sizes of foods. This evidence suggested that Noctiluca seemed to be a non-selective feeding organism. The sexual process in Noctiluca without its symbiont is similar manner as described by Zingmark (1970), who pointed out that the meiosis was occurred during the first 2 divisions of the diploid gametocyte mother cell. The assumption was based on the appearance of a tetrad which was maintained through the next five continuous division of the nuclei (Fig. 5.3-5.7). In natural samples, gametogenesis cells of Noctiluca with the green symbionts were also observed but the symbiotic algae have not been tranferred through this sexual process. They were transferred into daughter cells in asexual binarry fission. This study confirmed that green Noctiluca is basically non-selective heterotrophic organism. This dinoflagellate can live without the symbiotic green algae and the true colour of Noctiluca is pale-pink. The further study should be focused on the interaction between symbiotic green algae and Noctiluca. Acknowledgements The author would like to thank Dr.Yasuwo Fukuyo for his useful suggestion and comments and also to Miss Rujinard Sriwoon for her kind assistant in preparing the manuscript. References Charernphol, S. 1958. Preliminary study of discoloration of seawater in the Gulf of Thailand. In: Proceedings of the Ninth Pacific Congress, pp. 131-134. Enomoto, Y. 1956. On the occurrence and the food of Noctiluca scintillans (Macartney) in waters adjacent to the west coast of Kyushu, with special reference to the possibility of the damage caused to the fish eggs by that plankton. Bull.Jpn.Soc.Sci.Fish., 22: 82-88. Kimor, B. 1979. Predation by Noctiluca miliaris Souriray on Acartia tonsa Dana eggs in the inshore waters of Southern California. Limnol.Oceanogr., 24: 568-572. Okaichi, T. and Nishio, S. 1976. Identification of ammonia as the toxic principle of red tide of Noctiluca miliaris. Bull.Jpn.Soc.Sci.Fish., 23: 25-30. Okaichi, T., Nishio, S. and Imatomi, Y. 1983. Massculture of marine phytoflagellates, an approach to new sources of biologically active compounds. In: IUPAC Pesticide Chemistry, J. Miyamoto and P.C. Kearney (eds.). Pergamon Press,New York, pp.141-144.
Suvapepun, S. 1989. Occurrences of red tide in the Gulf of Thailand. In: Red Tide: Biology, Environmental Science and Toxicology, T. Okaichi and T. Nemoto (eds.). Elsevier, New York, pp. 41-44. Suvapepun, S. 1992. Trichodesmium blooms in the Gulf of Thailand. In: Marine Palegic Cyanobacteria: Trichodesmium and other Diazothophs, E.J. Carpenter et. al (eds.). Kluvers Academic Publishers, Netherland, pp. 343-348. Sweeney, B.M. 1971. Laboratory studies of a green Noctiluca from New Guinea. J.Phycol., 7: 53-58. Takayama, H. 1977. Culture of Noctiluca scintillans (Macartney). Bull.Plank.Soc.Jpn., 24: 83-86. Zigmark, R.G. 1970. Sexual reproduction in the dinoflagellate Noctiluca miliaris Souriray. J.Phycol., 6: 122-126.
Fig. 1 Food particles in vacuole of Noctiluca cells with no symbiotic green algae.
Fig. 2 Feeding process of Noctiluca scintillans on Dunaliella. viridis; 1) D. viridis were trapped by the motile tentacle of Noctiluca. 2) Increase of prey cells on tentacle. 3-4) The foods were brought to the cell surface near mouth groove. 5) Food paticles in vacuole of Noctiluca.
Fig. 3 Cannibal behavior of Noctiluca 1) beginning of cannibalic behavior; 2) 2) half of the prey cell was engulfed; 3) 3) completely ingested. t = tentacle.
Fig. 4. Noctiluca fed on suspended material in the culture medium.
Fig. 5. Gametogenesis in Noctiluca without its symbiont; 1) 1-nucleate stage; 2) 2-nucleate stage; 3) 4-nucleate stage; 4) 8-nucleate stage; 5) 16-nucleate stage; 6) 64-nucleate stage; 7) 128-nucleate stage; 8) the body of gametes on the surface of mother cell and its moving flagella (f) 9) swimming gamete with long flagella (f).
Fig. 6 The development from zygote to new Noctiluca; 1) a few hours after conjugation; 2) a few days later the nuclear mass has been seen clearly; 3) the new Noctiluca cell.