Micromonas pusilla (Prasinophyceae) as part of pico- and nanoplankton communities of the Barents Sea JAHN THRONDSEN and SVEIN KRISTIANSEN Throndsen, J . & Kristianaen. S . 1991: M i c r o m o m s p d / a (Prasinophyceae) a h part of pico- and nano- plankton communities in the Barents Sea. Pp. 201-207 in Sakshaug. E . . Hopkins. C. C. E. & Britsland, N . A . (eds.): Proceedings of the Pro Mare Symposium on Polar Marine Ecology, Trondheim, 12-16 May 1990. Polar Research 10(1). Micromonas pusilla (Butcher) Manton & Parkc appears to bc a prominent member of the Barcnts Sea picoplankton community as revealed by the serial dilution culture method. Cell numbcrs frcqucntly exceeded 10' cells I - ' , though they usually varied between 10' and 10'cells I - ' . A number of othcr identifed and unidentified taxa were recorded and quantified. Distribution relative to the marginal icc zonc 15 reported. Jahn Throndsen a n d Svein Kristiansen. Departmenr of Biology. Marine Botuny Division. University of O s l o . P . 0. Box 1069 Blindern, N-0316 Oslo 3, N o r w a y . Introduction Investigations on flagellates in the Barents Sea began with the work of Wulff (1916), who reported the presence of many species previously described from temperate waters. Meunier (1910), working in the rieighbouring area, the Kara Sea, likewise added to the knowledge of Arctic flagellate plankton and described new species from the area. Earlier investigations in polar oceans were restricted either to laboratory work on preserved material or to shipboard obser- vations of living material from net hauls. Recently, quantitative information on the smaller plankton of the Barents Sea was obtained by Reynolds (1973). who made direct counts, and Throndsen (1970), who used a dilution culture technique. Species information from Arctic areas has on the whole been obtained by electron micro- scopy studies on dried preparations of cell material fixed with osmic acid (e.g. Thomsen 1982, Manton et al. 1975, 1976). This method, however adequate for revealing submicroscopical details necessary for identification, gives very poor indications of cell numbers in the sea. The present survey elucidates the importance of euka- ryotic pico- and nanoplankton in the marginal ice zone in Arctic marine waters. The serial dilution culture method was applied i n order to give quan- titative as well as qualitative information. When first used in the Arctic in 1969 (Thrond- sen 1970), the dilution culture technique revealed the presence of a number of genera and species new to the waters off Spitsbergen and B j ~ r n ~ y a . Materials and methods Water samples were collected from stations at different distances from the ice edge (Fig. 1, Table 1) during Pro Mare cruises in the Barents Sea i n the spring (April) of 1986 and during the summers (June) of 1984 and 1987. The sampling device was assumed to be non- toxic; the effect of possible undetected toxicity was minimised by the relatively short handling time and the dilution of the samples into growth medium with chelating agents included. Serial dilution cultures (Throndsen 1978) were set up in Erd-Schreiber medium (S = 32.5) 1-2 hours after sampling. The cultures were kept in dim light at 2 4 ° C during the cruise and transportation and in the laboratory culture room at the Department of Biology, University of Oslo. The cultures were examined after 6-8 weeks by bright field and anoptral contrast microscopy, with additional observations by transmission elec- tron microscopy. The presence and absence of each species throughout the dilution culture series made it possible to estimate the cell numbers present in the original sample (MPN = Most Probable Num- ber). 202 J . Throndsen & S . Kristiansen 7 2 35 4 0 35 I \ 4 0 Fig. 1 . Map of the Barents Sea showing the location of the stations sampled for serial dilution cultures: solid circles = no ice; open circles = open pack ice: triangles = close pack ice. Results Picoplankton (1-2.5 pm) Micromonas pusilla (Butcher) Manton & Parke appears to be a prominent member of the pico- plankton community as revealed by the methods applied. Cell numbers frequently exceeded lo7 cells I - l , though they usually varied between 10' and 106cells I-' (Tables 2, 3). Out of 17 sta- tions sampled, M . pusilla was detected at all but three. Other eukaryotic picoplankton comprised green coccoids 1-1.5 pm, 2 pm, and 2.5-3 pm in size. These were not studied in further detail but probably included nonmotile M . pusilla, the newly described Bathycoccus prasinos (Eikrem & Throndsen 1990), and possibly also undescribed coccoid eukaryotic picoplankton species. Prokaryotic picoplankton were not prominent in the dilution cultures and are thus ignored in this paper. Nanoplankton (2.5-20 pm) Among the smaller nanoplankton (< 5 pm, Tab- les 2 , 3 ) identified, the five most abundant species were the prasinophycean Mantoniella squamata, the prymnesiophytes Dicrateria inornata and Imantonia rotunda, and the pedinelloid chry- sophyte Pseudopedinella tricostata. Table 1. Stations sampled for serial dilution cultures in the Barents Sea in 1984. 1986, and 1987 Year Date Ice condition Station Position Depth 1984 4 June 3 June 8 June 9 June 10 June 11 June 13 Aug open pack 670 no ice 676 no ice 721 no ice 723 no ice 744 no ice 847 open pack 733 77" 10". 33"B'E 76"40'N, 33"04'E 76"48'N. 33"07'E 76"lO'N. 35"00'E 76"32'N. 45.00'E 75"45'N, 45"00'E 76"40'N. 33"04'E 5 m 25 m 0.5,10.20.30,50.75 m 10 m 5.15.75 m 10 m 10 m 1986 10 April open pack 018 75"29'N. 32"06'E 10 m 15 April no ice 028 74"00'N. 27"Il'E 10 m 16 April open pack 03 1 74"5U'N. 27"46'E 10 m 17 April close pack 037 74-54! N , 32"59'E ice 19 April close pack 043 75"54'N. 30"45'E ice 20 April close pack H- 1 21 April no ice 052 74'33". 27"50'E 10 m, bloom 75-26". 34"15'E 10.30 m under ice 1987 2 June no ice 945 74-11". 28"00'E 10 m 3 June no ice 96 1 75"55'N. 30"30'E 10.30 m 8 June no ice 994 74"30'N. 3 I"3 1 'E 10.60 m Nanoplankton communities of the Barents Sea 203 Table 2. Occurrence of Micromonas pusilla and selected groups of plankton on stations sampled in the Barents Sea in Junc 1986. MPN in lo’ ccllsl-’. - no cells recorded. STATIONS Species/groups 670 733 676 721 723 744 Ice condition open pack ice no ice Sampling depth 5 rn 5 m 2 5 m Om 10 m 10 m Micromonas pusilla Other eukaryotic picoplankton Nanoplankton 4 pn Nanoplankton > 5 pm Diatoms Heterotrophic flagellates >24 000 78 - 68 170 270 31 48 0 . 4 94 120.2 73.2 2 0 56 0.8 40 19 72.2 0.2 20 - - 18 - - 240 920 260 260 35 - - - - 0 . 4 6 Table 3. Occurrence of Micromonos pusilla and selected groups of plankton In the Barents Sea In Apnl 1986. MPN i n 10‘ cells I - ] , - no cells recorded. STATIONS 037 043 H- 1 018 031 028 052 Ice condition close pack ice open pack ice no ice ~~~~~ ~~ I .8 Other eukar. picoplkt. - - 110 210 60 6.8 0.8 Micromonas pusilla 2 - 0.2 40 I3 OOO - Nanoplankton < 5 pm - 2.2 22 40 - 55 120 Nanoplankton > 5 pm - - 0.2 0.2 20 Heterotroph. flags 176 22 - - Diatoms 19 500 24 300 84 100 1830 142 254 - - - - - Mantoniella squamata (Manton & Parke) Desi- kachary (3-5 pm) belongs to the smaller nano- plankton. It was recorded on three occasions only. Other prasinophycean flagellate species, e.g. of Pyramimonas, were even more scarce in the dilu- tion cultures. Non-motile stages of Pterosperma, Halosphaera and Pachysphaera were encountered in small numbers only. Dicrateria inornata Parke (3-5.5 pm) and Iman- tonia rotunda Reynolds (2-4 pm) are almost indis- tinguishable in the light microscope, and for this reason they were referred to as Dicrateria inornatallmantonia rotunda. Most often samples studied in the electron microscope appeared to contain Imantonia rotunda which is characterised by fine oval-circular, cartwheel-patterned organic scales covering the cell surface. Dicrateria inor- nata is naked and information from direct EM preparations was rather limited. Larger nanoplankton (> 5 pm) (Tables 2 , 3) was usually dominated by chryso- or crypto- phytes. Pseudopedinella pyriformis N . Carter (5- 8 pm) was the most common chrysophyte ident- ified, the smaller P . tricostata (Rouchijajnen) Thomsen (4-5.5 pm) was less common, but re- corded both in 1984 and 1987. Motile cells of the genus Phaeocystis (4.5-8 pm) were also occasionally significant. A new species “Arctic flag sp.#” was probably the most common taxon among the larger nano- plankton. The species has flattened cells 9-12 pm long, with two ventrally inserted flagella, one pointing forwards, the other directed posteriorly. The single chloroplast is golden brown. A more extensive description of the species will be pub- lished elsewhere. Cryptophycean species in the size range 1C- 20pm were frequently present. Species of the genera Isoselmis, Plagioselmis and Chroomonas covered the whole nanoplankton size spectrum, whereas specimens which were tentatively ident- ified as Cryptomonas prora Conrad & Kufferath were in the upper size range (18-21 pm). This species has been reported to bloom during winter 'E pue sqqeL uI .a.a uoiyueldoueu iaqio woij palwedas pue sdnoJ% se 01 paiiajai kluo Allensn aJaM smoieip pue saie[[a%eu siqdoiioiaiaH .payyuap! sezn iaqsing sua~sauun.iq 'H Lluo sapads s!wias!wa~ aqi 30 '(pgji qieiajjny 78 peiuo3) wn!%pg ui siaiem qslyseiq uI (~,,S.O-) as! aqi iapun punoj uaaq aAeq 01 uaha pue 3 I 3 d Nunoplankton communities of the Barenrs Sea 205 have been used to allow for a better estimate of these high M . pusilla populations. Close to the ice edge (Station 031), MPN up to 13 million cells I-' was recorded in open pack ice in April 1986 (Table 3). For the stations in oceanic ice-free waters, the Micromonas numbers varied between lo6 and lo3 cells I - ' , except for Station 028 where it was not recorded. The lowest recorded concentrations of M . p u s - illa were near the limit of detection (180 cells I - ' ) and were found at 20 m depth at Station 721, 15- , 20 nm south of the ice edge in 1984 and at 30 m under the ice at Station H-1 in 1986 (Table 4). Depth distribution (Station 721) Information on depth distribution is sparse. The MPN for Micromonas pusilla and four groups of pico- and nanoplankton from surface to 75m depth at Station 721 (1984, Table 5) may, however, reflect a common situation; the diatoms had their highest numbers in the surface layer and at 20 m. M . pwilla was likewise numerous at the surface, but its deeper maximum was at 50 metres. At Station 733 three depths were sampled show- ing the highest concentrations of Micromonas at 5 m, slightly lower at 15, and lacking at 75 m. At Station 721 dilution cultures from 5 depths (Table 5) showed that the dominating populations of viable pico- and nanoplankton were confined to the upper 30 m. The population at 75 m con- sisted of non-motile picoplankton and nano- plankton flagellates: Mantoniella squamata, Pseu- dopedinella pyriformis and the heterotrophic Pseudobodo minimus Ruinen, as well as an unidentified heterotrophic Gymnodinium species. M . pusilla was most numerous at the surface, but the concentration was only 7 x lo4 cells I-'. The concentration of Pseudopedinella pyri- formis varied from 6 lo3 cells I - ' at the surface to 4 . lo2 cells 1-' at 75 m, with a maximum of 45 lo3 at 30 m. Occurrence of other flagellates Pseudopedinella species were most common in summer samples, the highest MPN were 45 lo3 cells.1-' (Station 721, 3 0 m depth) for P . pyri- formis whereas P. tricostata never exceeded 3.7 lo3 cells I - ' . P . pyriformis was, however, found from the surface to 75 m depth. Heterotrophic flagellates of nanoplankton size were present at most stations. MPN varied from the detection limit 180cells I - ' to 2.1 * lo5 cells I - ' . The highest cell number recorded was for Par- aphysomonas at Station 994, 60 m in June 1987, whereas 1.7 - lo5 cells I - ' of an unknown species was estimated from an under-ice sample at Station 037. Discussion The SDC method The serial dilution culture method is known to be selective (Throndsen 1978), and other species than those recorded may certainly also have been present in the samples. Lack of viability of the specimens introduced to the dilution tubes, and competitive relations in the single tube, may have reduced the number of tubes with detectable growth of an otherwise culturable species. The culture conditions and the medium used will further influence growth, and hence the MPN estimates (see e.g. Furuya & Marumo 1983, Jochem 1990). The presence of the species recorded, however, cannot be disputed, and they certainly belong to the viable part of the plankton community. Heterotrophic species will only grow provided adequate food organisms or compounds are present, and there is certainly a lack in regis- tration especially of heterotrophic dinoflagellates. Technically, correct MPN estimates for cul- turable species will be impossible if their original concentration is too high compared with the dilu- tions used. The five step dilution used ended with an inoculum of 0.1 pI of the original sample. During the work in the Barents Sea this caused limitations in some of the estimates of the Mic- romonas pusilla population; MPN estimates had to be given as > 24 million cells 1-'. For larger species such high numbers of cells would have caused discoloration of the water and hence given visual advice regarding the number of dilution steps. The MPN estimates could be compared with direct counts at Station 994 (T. F. Thingstad pers. comm.). The MPN for Micromonas at 10 m depth was higher than 2.4. lo7 cells l-', whereas direct counts for picoplankton (not identified to species) gave 1.5 - lo7 cells I-'. This indicates that Mic- romonas probably was the major if not the only species in the group. The difference in cell number 206 J . Throndsen & S . Kristiansen estimates is an acceptable discrepancy considering the methods used. At 60rn the difference was significant, MPN estimate for Micrornonas was 1.7. lo6 cellsl-* whereas the counts gave neg- ligible numbers o n l y . Phaeocystis MPN estimates were related t o colonies (which could contain a variable number of cells) and hence gave lower numbers than the direct cell counts. Occurrence related to the ice Micromonas pusilla was apparently not par- ticularly favoured by the conditions in or very close to the ice, though fairly high cell numbers were recorded also at the ice edge (Fig. 1 , Table 4), the latter being in accordance with the tol- erance of this species for low salinities as well as low temperatures (Throndsen 1976; Throndsen & Heimdal 1976; Jochem 1990). The low surface to volume relation is likely to give the pico- plankton species their best competitive advantage under oligotrophic conditions, and it is therefore not surprising to see that diatoms (mostly nan- oplanktonic species were recorded by the method applied) rather than picoplankton were dom- inating in the ice underside samples. The oppor- tunistic M . pusilla, however. was very abundant in the surface water of the marginal ice zone. Information on M . pusilla from other polar oceans is mostly restricted to identification in direct EM preparations, and with no real evidence for cell concentrations. Also for eukaryotic pico- plankton in general the quantitative information from these areas is very sparse. From the Tromso area, which is north of the Arctic circle and hence is exposed to "polar" light conditions, M . pusilla proved to be present in the sea during all the seasons, but with its highest numbers in summer (Throndsen & Heimdal 1976). Depth distribution Micromonas pusilla is known to have a low light saturation level for growth (Throndsen 1976). and its depth distribution in subtropical areas is extensive: viable cells are found down to 500 m i n the Bay of Biscay (Manton & Parke 1960) and 600111 off the coast of Japan (Kuroshio area. Throndsen 1983). Hence the presence of M . pus- illa at 75 rn in the Barents Sea is to be expected. Its distribution in the deeper water masses, including productive areas at high latitudes, further con- firms its ability for survival and growth. Studying the subsurface chlorophyll maximum in the western North Pacific Ocean, Furuya & Marumo (1983) found Micromonas (probably M . pusilla) to be the most abundant genus in this layer. In the Gulf of Panama M . pusilla appeared to follow the general distribution of chlorophyll from 10 to 100m depth; the deeper maximum here was at 75 m (Throndsen 1976). Picoplankton importance in polar waters Though Micromonas pusilla and other eukaryotic picoplankton species have been reported from both Arctic (Throndsen 1970; Thomsen 1982) and Antarctic waters (H. A. Thomsen pers. cornm.), little is known about their importance relative to the prokaryotic picoplankton in these areas. Cyanobacteria are frequently recorded by the dilution culture method e.g. in coastal areas of the temperate zone, but they were not common in the present samples. Based on the methods known today, future efforts i n this field should include an integrated use of serial dilution cultures, direct EM prep- arations, epifluorescence counts, size fractionated carbon uptake measurements, and laboratory growth and pigment studies. Acknowledgements. - T h e authors thank the officers and crew ance. This project was supported by The Norwegian Research Program for Marine Arctic Ecology (Pro Mare). of R / V G 0 SARS. K/V ANDENES and K / V SENJA for their assist- References Conrad. W . & Kufferath. H . 1954: Rechcrches sur les eaux saumatres des environs de Lilloo. 2 . Partie descriptive. Mem. Insr. R . Sci. Not. Belg. 127. 1-346. Eikrem. W. & Throndsen. J . 1990: The ultrastructure of Bathy- coccus gen. nov. and B. prasinos sp. nov.. a nonmotile picoplanktonic alga (Chlorophyta. Prasinophyceae) from the Mediterranean and Atlantic Phycologia 29. 344350. Furuya. K. & Marumo, R. 1983: The structure of the phyto- plankton community in the subsurface chlorophyll maxima In the western North Pacific Ocean. 1. Plankton Res. 5 . 393- 406. Jochcm. F. J . 1990: On the seasonal occurrence of autotrophic naked nanoflagellates in Kiel Bight. Wcstern Baltic. Estrtnr. Coast. Shelf Sci. 3 1 . 189-202. Manton. I . , & Parke. M. 1960: Further observations on small grcen flagellates with special reference to possible relatives of Chromulina pusilla Butcher. J . Mar. Biol. Ass. U . K . 39, 275-298. Manton. I . . Sutherland. J . & Leadbeater. B S. C. 1975: Four new species of choanoflagellates from Arctic Canada. Proc. R . Soc. London, Ser. B 189. 15-27. Nanoplankton communities of the Barents Sea 207 Manton. I . . Sutherland. J . & Leadbeater. B. S. C. 1976: Further observations on the fine structure of collared flagellates (Choanoflagellata) from Arctic Canada and West Greenland. Can. J . Bot. 54, 1932-1955. Meunier, A . 1910. Microplankton des mers de Barents et de Kara. Duc d'Orl6ans Campagne Arctique de 1907, Bruxelles. Reynolds, N . 1973: The estimation of the abundance of ultra- plankton. Br. Phycol. J . 8. 135-146. Thomsen, H. A. 1982: Planktonic choanoflagellates from Disko Bugt. West Greenland, with a survey of the marine nano- plankton of the area. Meddr. G r ~ n l a n d . Biosci. 8 , 1-36. Throndsen. J . 1970: Flagellates from Arctic waters. N y t t Mag. Bot. 17. 49-57. Throndsen. J. 1976. Occurrence and productivity of small mar- ine flagellates. Norw. J . Bot. 23. 269-293. 355 pp. Throndsen, J . & Heimdal. B. R. 1976: Primary production, phytoplankton and light in Straumsbukta near Tromsb. Astarte 9 , 5 1 4 0 . Throndsen. J . 1978: The dilution culture method. Pp. 218- 224 in Sournia. A . (ed.): Phytoplankton Manual. UNESCO Monographs on Oceanographic Methodology, No. 6. Paris. Throndsen, J. 1983: Ultra- and nanoplankton flagellates from coastal waters of southern Honshu and Kyushu, Japan (including some results from the western part of the Kuroshio off Honshu). Working party on taxonomv in the Akashiwo Mondai Kenkyukai fishing ground preservation dillision, Research department, Fisheries Agency, Japan. 62 pp. W u l f f , A . 1916: Ueber das Kleinplankton der Barentssee. Arb. Dt. Wiss. Kommn Int. Meeresforsch. A . A w Dem Lab- oratorium Fur lnternationale Meeresforschung in Kiel. Biol. Abt. No. 28, 95-125.