Temporal Change of Clustered Distribution of Planktonic Ciliates in Toyama Bay in Summers of 1989 and 1990

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1 Journal of Oceanography, Vol. 53, pp. 35 to Temporal Change of Clustered Distribution of Planktonic Ciliates in Toyama Bay in Summers of 1989 and 1990 TOSHIKAZU SUZUKI* and AKIRA TANIGUCHI Faculty of Agriculture, Tohoku University, Sendai 981, Japan (Received 29 January 1996; in revised form 21 May 1996; accepted 21 May 1996) Temporal change of clustered distribution in vertical profiles of three nutritional groups of planktonic ciliates, e.g. heterotrophic naked ciliates, mixotrophic naked ciliates and heterotrophic loricated ciliates, was investigated by following a drifting buoy in Toyama Bay on the Japan Sea coast of central Japan in summers of 1989 and Clustered distribution, represented as the mode of population density in the vertical plane, occurred mainly in the oligotrophic upper layer (0 50 m depth) above the subsurface chlorophylla maximum layer. Its clustered degree was stronger when the mode of population density in the vertical plane was formed at shallower depth, while its longevity was shorter as mentioned above. Vertical distribution of ciliates during summer in Toyama Bay is characterized by ephemeral clustered distribution, or in other wards, by rapid alternations of appearance and disappearance of the clustered distribution. Keywords: Planktonic ciliates, vertical profile, clustered distribution, temporal change, Toyama Bay. 1. Introduction Planktonic ciliates occur commonly and abundantly in the surface layer of the world s oceans. Their growth rates are very high under suitable conditions, e.g. generation time is as short as 6 h. in Strombidium sp. (Paranjape and Gold, 1982; Laybourn-Parry, 1984), 6 14 h. in Helicostomella subulata (Paranjape, 1980) and h. in Eutintinnus lususundae and Eutintinnus tubulosus (Taniguchi and Kawakami, 1983; Suzuki and Taniguchi, 1993). Consequent rapid increase in numerical abundance at a certain depth, related to preferential conditions, can modify their vertical distributional pattern as observed in phytoplankton (Sournia, 1974). However, the spatio-temporal change of vertical distribution of ciliates in nature has scarcely been investigated, except in the works of Dale (1987) and Stoecker et al. (1989) which examined diel vertical migration. In this study we examine the vertical distribution of ciliates in summer in Toyama Bay on the Japan Sea coast of central Honshu, Japan, to clarify the temporal change of the clustered distribution. It is important to eliminate the influence of change of water parcel during this kind of investigation. A floating buoy experiment is ideal in tracing a particular water parcel which moves horizontally along a water current (McManus and Fuhrman, 1986; Heinbokel, 1987; Stoecker et al., 1989). We sampled the vertical profiles of ciliates by following the *Present address: Faculty of Fisheries, Nagasaki University, Bunkyo-cho 1-14, Nagasaki 852, Japan. floating buoy deployed in the upper layer of a vertically stratified water column where mixing and turbulence of water is small. 2. Materials and Methods A floating buoy attached with a parachute drogue was deployed twice near Stn. A in Toyama Bay, drifting during July 1989 and during 3 6 August 1990, where the OTEC (Ocean Thermal Energy Conversion) barge was anchored (Fig. 1). During these investigations the barge was continuously sprinkling a mixture of deep water and surface water at a rate of 900 l s 1 (Suzuki and Taniguchi, 1993). The time duration and drift speed of the buoy were respectively 31 h. and cm s 1 in 1989 and 60 h. and 1 8 cm s 1 in The parachute, 4.2 m in diameter, was set at the depth of 17 m in 1989 and 20 m in 1990, where the water density was the same as the density of the water sprinkled by the barge. Water samples were taken three or four times a day at 13 depth layers in the top 75 m water column in 1989 and 7 depth layers in the top 100 m in 1990 from a research vessel following the buoy. The samples were immediately fixed by adding borax-buffered formaldehyde at a final concentration of 2%. The samples were stored in a dark, cool place until later microscopic examination in order to prevent the possible degradation of chlorophyll pigments of mixotrophic ciliates (Suzuki and Taniguchi, 1993). Enumeration of the individual number of ciliates was achieved for a 100 ml aliquot under an Olympus IMT-2 epifluorescence inverted microscope at 200X magnification by following the Utermöhl Copyright Oceanographic Society of Japan. 35

2 method (Hasle, 1978). Mixotrophic naked ciliates were identified by possession of chloroplasts which emit bright red fluorescence under a B-excitation light, being different from the spotted and faded red emission of the ingested picoplankton by heterotrophic ciliates. Since the numerical abundances of individual species were not always large enough for statistical analysis, all the ciliate species occurred were categorized into three groups, i.e. mixotrophic naked ciliates, heterotrophic naked ciliates and heterotrophic loricated ciliates (tintinnids) from the viewpoint of their trophic modes (Stoecker et al., 1989; Suzuki and Taniguchi, 1993). To evaluate clustered distribution in the vertical plane, the clustered degree (CD; cells m 1 ) or mean gradient of population size around the depth where the mode of population density occurred is calculated as follows, CD = [(Cm Cu)/(Dm Du) + (Cm Cd)/(Dd Dm)]/2, where Cm, Cu and Cd are respectively numerical abundances of ciliates at the depth where the mode of population density occurred (D mode ), at upper neighboring sampling layer and at deeper neighboring sampling layer. Dm, Du and Dd are depth in meters of D mode and its neighboring upper and deeper sampling layers. When D mode occurred at 0 m or at the deepest sampling depth, CD was expressed as a single gradient. The rate of temporal change (TC; cells h 1 ) or mean temporal gradient around the time when the mode of population density was formed is also calculated in similar way as follows, TC = [(Cm Cp)/(Tm Tp) + (Cm Cn)/(Tn Tm)]/2, where Cm is abundance at D mode and Cp and Cn are abundances at the same depth at just previous and just next sampling times respectively. (Tm Tp) or (Tn Tm) are intervals in hours between the time when the mode of population density was formed and its previous or next sampling time. When Tm was the initial or the final time of a series of samplings, TC was expressed as a single rate of change. Fig. 1. Tracks of a drifting buoy and sampling stations in Toyama Bay. Samples were taken during July, 1989 (Stns. M1 to M6) and during 3 6 August, 1990 (Stns. Y1 to Y9). 3. Results Toyama Bay is under strong influence of the intrusion of the Tsushima Warm Current in the surface layer, which Table 1. Average abundances of three groups of ciliates. HNC: heterotrophic naked ciliates, MNC: mixotrophic naked ciliates and HLC: heterotrophic loricated ciliates. Location Season/time Abundance (cells l 1 ) Reference HNC MNC HLC North-western subtropical Pacific May, Suzuki (1994) East China Sea, Basin area August, Ota (1995) Onagawa Bay July, Iwamatsu (1989) Toyama Bay July, This study Toyama Bay August, This study 36 T. Suzuki and A. Taniguchi

3 flows counterclockwise along the coast of the bay during summer and autumn. During these seasons, surface temperature increases and strong thermal stratification is established at depths shallower than 100 m (Hirakawa et al., 1990). Such a hydrographic condition makes surface water oligotrophic, even near the coast. Ciliate abundance observed in this study was between the levels in other neritic waters and subtropical open waters (Table 1). In this investigation we could not observe any consistent increase in density of ciliate populations due to sprinkled deep-sea water, although this was originally expected. No increase was detected for phytoplankton standing crops, either, indicating that the amount of water sprinkled was too small to enrich and modify the natural planktonic populations in the area investigated (Iseki et al., 1994) July 1989 Data on environmental parameters during the drifting experiments have been provided by the Japan Sea National Fisheries Research Institute. The data show that water temperature was C at 0 m and decreased linearly with depth to C at 75 m. Salinity increased with depth from PSU at 0 m to PSU at 75 m. Chlorophyll-a concentration was as low as 0.1 µg l 1 at 0 m forming a subsurface maximum layer of µg l 1 at m. Such vertical profiles of temperature, salinity and chlorophyll-a remained unchanged during the drifting experiments of 31 hs. Heterotrophic naked ciliates showed more or less homogeneous vertical distribution between 0 m and 75 m, although two sporadic peaks were observed at 5 m at Stns. M2 (550 cells l 1 ) and M4 (820 cells l 1 ) (Fig. 2a). The mode of population density occurred in the shallower layer (0 5 m), except for Stn. M1 (75 m depth). The change of D mode was very small after the sampling at Stn. M2. Mixotrophic naked ciliates concentrated to cells l 1 in a subsurface layer between 20 m and 50 m at every station. Large abundance of cells l 1 was also observed at the surface at Stns. M2, M4 and M6 (Fig. 2b). D mode was formed in the mid layer of m depth except for Stns. M4 and M6. Heterotrophic loricated ciliates concentrated slightly to cells l 1 in a subsurface layer of m at every station (Fig. 2c). At the surface at Stns. M2, M3 and M6, a marked peak of cells l 1 was also observed. D mode was at the surface, except for Stns. M1 and M August 1990 Water temperature decreased linearly with depth from C at 0 m to C at 100 m depth. Salinity increased from PSU at 0 m to at 100 m. A subsurface chlorophyll-a maximum layer of µg l 1 was observed at m. Vertical profiles of these environmental factors were constant during the 60 h. of the experiment. Fig. 2. Temporal changes in vertical distribution (cells l 1 ) of the heterotrophic naked ciliates (a), mixotrophic naked ciliates (b) and heterotrophic loricated ciliates (c) observed in July Arrow indicates vertical mode depth. Temporal Change of Clustered Distribution of Planktonic Ciliates 37

4 Heterotrophic naked ciliates were abundant in the subsurface layer of m, forming broad peaks of over 300 cells l 1 at m at Stn. Y1 and at m at Stns. Y6 and Y7 (Fig. 3a). Their vertical gradient was small. D mode was in the m depth layer. Mixotrophic naked ciliates were abundant in the top 70 m (300 cells l 1 ) forming some sporadic peaks at 30 m at Stn. Y1 (740 cells l 1 ), 0 m at Stn. Y5 (1480 cells l 1 ) and 20 m at Stn. Y8 (760 cells l 1 ). Below 70 m, they decreased rapidly to smaller than 50 cells l 1 at 100 m, except for Stn. Y1, where the abundance was over 500 cells l 1 (Fig. 3b). Their vertical gradient was larger than that of heterotrophic naked ciliates. D mode was in the 0 70 m depth layer. Heterotrophic loricated ciliates showed a relatively homogeneous distribution with low abundance in the entire water column, while three small peaks were observed at 70 m at Stns. Y3 (260 cells l 1 ) and Y5 (160 cells l 1 ) and at 100 m at Stn. Y1 (180 cells l 1 ) (Fig. 3c). D mode tended to be in shallower or deeper layer. Its variation is largest among the three ciliate groups. Continuity of D mode was not observed. When clustered degree of ciliates in the vertical plane around D mode is plotted against D mode, an inverse relationship is detected (Fig. 4). In the present investigations, the detected relationship above 50 m depth is reliable since the samples were taken at the same intervals above 50 m. As a whole, strong CD, e.g., those of mixotrophic naked ciliates in 1989 and 1990 and of heterotrophic naked ciliates and heterotrophic loricated ciliates in 1989, occurred in shal- Fig. 3. Temporal changes in vertical distribution (cells l 1 ) of the heterotrophic naked ciliates (a), mixotrophic naked ciliates (b) and heterotrophic loricated ciliates (c) observed during 3 6 August Arrow indicates vertical mode depth. Fig. 4. Relationship between D mode (m) (depth where mode of population density occurred) and clustered degree of ciliates (cells m 1 ) in vertical plane around D mode. Definition of the clustered degree is given in text. Filled circle: heterotrophic naked ciliates in 1989, filled triangle: mixotrophic naked ciliates in 1989, filled square: heterotrophic loricated ciliates in 1989, open circle: heterotrophic naked ciliates in 1990, open triangle: mixotrophic naked ciliates in 1990 and open square: heterotrophic loricated ciliates in T. Suzuki and A. Taniguchi

5 lower layer, while weak CD, such as those of heterotrophic naked ciliates and heterotrophic loricated ciliates in 1990, were in both shallower and deeper layers. 4. Discussion Loss of cells due to fixation and preservation is a serious problem in determining ciliate abundance. Although formaldehyde fixation is the most appropriate method for preserving the fluorescence of chloroplasts sequestered in mixotrophic naked ciliates, it results in a lower cell count than fixation with acid Lugol s solution or Bouin s solution (Stoecker et al., 1994). Ciliate abundance determined in this study, therefore, might be more or less underestimated. However, its relative value is reliable due to the consistent employment of this fixation procedure throughout this study. As mentioned above, no consistent growth of ciliate populations was promoted by sprinkling deep-sea water at the rate used. This condition, however, favors the analysis of diel change in vertical distribution. At first we tried to detect evidence of diel vertical migration, but no positive evidence was found at all. During the course of these experiments we perceived the ephemeral nature of the clustered distribution of ciliates. If cells divide at a particular time at a certain depth where environmental conditions are preferential, it can be erroneously concluded that they had migrated there actively (Sournia, 1974). Changes of D mode observed in three ciliate groups in Toyama Bay were frequently very large. D mode of heterotrophic naked ciliates and mixotrophic naked ciliates in 1989 (Fig. 2) and those of mixotrophic naked ciliates and heterotrophic loricated ciliates in 1990 (Fig. 3) shifted more than 50 m during the sampling intervals of 6 12 h. Such a large change of D mode could not be achieved by synchronized swimming of ciliates at their general swimming speed of ca. 1 mm s 1 (Sleigh and Blake, 1977). One possible cause was a combination of increase in population density at new D mode and decrease at previous D mode. This means that the vertically clustered distribution of ciliates is discontinuous and ephemeral, being less than several hours to several days in Toyama Bay in summer. Frequent appearance and disappearance of the vertically clustered distribution might be inherent in ciliate populations in oligotrophic waters. In Toyama Bay in summer, water column is well stratified and temperature is higher in the upper layer, e.g. 27 C at 0 m. In spite of low chlorophyll-a concentration, this high temperature in the upper layer could promote the growth of ciliates (Finlay, 1977; Wickham and Lynn, 1990) and may be the reason for the occurrence of strong clustered degree in the vertical plane around D mode at shallower depth (Fig. 4). The enormous increase of mixotrophic naked ciliates at 0 m at Stn. Y5 in 1990, exceptionally can not be explained by this reasoning. A different water mass, bringing abundant mixotrophic ones, might temporarily intrude at the surface. Clustered degree of ciliates in the vertical plane around D mode was directly proportional to the rate of temporal change around the time when the mode of population density was formed (Fig. 5). Although CD and TC were low especially for heterotrophic naked ciliates and heterotrophic loricated ciliates in most cases in 1990, the regression coefficient of TC to CD was significant: TC = CD, r = 0.78, P < This significant correlation means that intensive concentration of ciliates in distribution is ephemeral compared with homogeneous distributional patterns. Relationships in abundance between preys and ciliates and/ or between ciliates and predators might not be stable, while the relationships could not be determined due to lack of the data on abundances of pico- and nanoplankton as preys and of netzooplankton as predators in this study. The mode of population density in the vertical plane occurred mostly in the oligotrophic upper layer for all of three ciliate groups, being shallower than the subsurface chlorophyll-a maximum layer. Its clustered degree tended to be stronger in shallower layer, while its longevity became shorter as mentioned above. In conclusion, vertical profiles of ciliates are characterized as rapid alternation of appearance and disappearance of clustered distribution in the surface oligotrophic waters like Toyama Bay water in summer. This suggests that Fig. 5. Relationship between vertical clustered degree of ciliates (cells m 1 ) in vertical plane around D mode and the temporal change (cells h 1 ) around the time when mode of population density was formed. Definitions of the clustered degree and temporal change are given in text. Filled circle: heterotrophic naked ciliates in 1989, filled triangle: mixotrophic naked ciliates in 1989, filled square: heterotrophic loricated ciliates in 1989, open circle: heterotrophic naked ciliates in 1990, open triangle: mixotrophic naked ciliates in 1990 and open square: heterotrophic loricated ciliates in Temporal Change of Clustered Distribution of Planktonic Ciliates 39

6 the detection of vertical migration of the ciliate populations in the oligotrophic waters is not easy task. Acknowledgements We thank the staffs of Japan Sea National Fisheries Research Institute for kind permission to cite the environmental data. We also thank Drs. K. Hirakawa, T. Ikeda, K. Iseki, H. Nagata and T. Odate for their kind collaboration in the field work and also for encouraging discussion of this topic. This study was mainly supported by the Special Coordination Fund for Promoting Science and Technology of the Japanese Science and Technology Agency. References Dale, T. (1987): Diel vertical distribution of planktonic ciliates in Lindåspollene, western Norway. Mar. Microb. Food Webs, 2, Finlay, B. J. (1977): The dependence of reproductive rate on cell size and temperature in freshwater ciliated protozoa: feeding rates and their ecological significance. Microb. Ecol., 6, Hasle, G. R. (1978): The inverted-microscope method. p In Phytoplankton Manual, ed. by A. Sournia, UNESCO, Paris. Heinbokel, J. F. (1987): Diel periodicities and rates of reproduction in natural populations of tintinnines in the oligotrophic waters off Hawaii, September Mar. Microb. Food Webs, 2, Hirakawa, K., T. Ikeda and N. Kajihara (1990): Vertical distribution of zooplankton in Toyama Bay, southern Japan Sea, with special reference to Copepoda. Bull. Plankton Soc. Japan, 37, Iseki, K., H. Nagata, K. Furuya, T. Odate and A. Kawamura (1994): Effect of artificial upwelling on primary production in Toyama Bay, Japan. p In MIFS 94: Proceedings of the 1994 MIE International Forum & Symposium on Global Environment and Friendly Energy Technology; March 22 25, 1994, Tsu, Mie, Japan, ed. by Y. Shimizu, S. Kato and M. Hoki, Mie Academic Press, Mie. Iwamatsu, M. (1989): Abundance of mixotrophic and heterotrophic planktonic ciliates in Onagawa Bay. Graduate Thesis, Tohoku Univ., Sendai, 117 pp. (in Japanese with English abstract). Laybourn-Parry, J. (1984): A Functional Biology of Free-Living Protozoa. Univ. Calif. Press, Berkley & Los Angels, 218 pp. McManus, G. B. and J. A. Fuhrman (1986): Photosynthetic pigments in the ciliate Laboea strobila from Long Island Sound, USA. J. Plankton Res., 8, Ota, T. (1995): Higashishinakai ni okeru Bisho Dobutsu Purankuton tokuni Senmotyu Purankuton no Genzonryo to Seisanryo (Biomass and production of microzooplankton in the East China Sea with special reference to planktonic ciliates). Master s Thesis, Tohoku Univ., Sendai (in Japanese with English abstract). Paranjape, M. A. (1980): Occurrence and significance of resting cysts in a hyaline tintinnid, Helicostomella subulata. J. Exp. Mar. Biol. Ecol., 48, Paranjape, M. A. and K. Gold (1982): Cultivation of marine pelagic protozoa. Ann. Inst. Oceanogr. Paris, 58, Sleigh, M. A. and J. R. Blake (1977): Method of ciliary production and their size limitations. p In Scale Effects in Animal Locomotion, ed. by T. J. Pedley, Academic Press, London. Sournia, A. (1974): Circadian periodicities in natural populations of marine phytoplankton. Adv. Mar. Biol., 12, Stoecker, D. K., A. Taniguchi and A. E. Michaels (1989): Abundance of autotrophic, mixotrophic and heterotrophic ciliates in shelf and slope waters. Mar. Ecol. Prog. Ser., 50, Stoecker, D. K., D. J. Gifford and M. Putt (1994): Preservation of marine planktonic ciliates: losses and cell shrinkage during fixation. Mar. Ecol. Prog. Ser., 110, Suzuki, T. (1994): Production of planktonic ciliates and their role in partitioning of carbon flux in the western subarctic and subtropical Pacific Ocean. Ph.D. Thesis, Sendai, Tohoku Univ., 203 pp. Suzuki, T. and A. Taniguchi (1993): Successional sequence of ciliates in surface water after a pulsed addition of deep water. Bull. Plankton Soc. Japan, 40, Taniguchi, A. and R. Kawakami (1983): Growth rates of ciliates Eutintinnus lususundae and Favella taraikaensis observed in the laboratory culture experiments. Bull. Plankton Soc. Japan, 30, Wickham, S. A. and D. H. Lynn (1990): Relations between growth rate, cell size and DNA content in colpodean ciliates (Ciliophora: Colpodea). Europ. J. Protistol., 25, T. Suzuki and A. Taniguchi