'Instituto de Oceanografia, Faculdade de Ciencias da Universidade de Lisboa, 1749-016 Lisboa, Portugal ..... Annual Review, UCL Press, J1, pp. 97-161 (1997).
133
ON A L1NGULODINWM POL YEDRUM BLOOM IN SEnJBAL BAY, PORTUGAL Ana Amorim·, Ana Sofia Palma··, Maria Ant6nia Sampayo·· and Maria Teresa Moita" 'Instituto de Oceanografia, Faculdade de Ciencias da Universidade de Lisboa, 1749-0 16 Lisboa, Portugal .. Instituto de Investiga9!O das Pescas e do Mar (lPIMAR), Av. Bras ilia, 1449-006 Lisboa, Portugal
ABSTRACT On II September 1996, the HAB monitoring program detected the presence of Lingulod inium po/yedrum in Serubal Bay. One week later, a brownish discoloration of seawater was noti ced and t he species reac hed concentrations of 7xl 0 5 cells.rl . The bloom lasted 10 days and the accompanying assemb lage was dominated by Prorocenlrum micans. A hydrograph ic/ phytoplankton cross-shelf section, carried out in the Bay du ring the bloom period, revealed cond itions of strati fication, with a vertica.l gradient of 2.5°C between 10 and 30m depth. L. polyedrum was recorded down to 20m, with maximum densities above the thermocline, at 5m depth. One week after bloom detection, the encystment process was already occurring. L. polyedrum cysts were observed in the water samples, representing ca 0 .1 % of the spec ies counts. Just after the bloom, and in the followi ng months, sediment samp les co llected south of Setu bal bay, showed an increase in cysts of L. polyedrum with apparently viable cell content. Th is bloom event was not associated with shell fish toxicity or any other harmfu l effects. INT RODUCTION
Lirzgu /odini um p o /yedrum (Stein) Dodge is an autotroph ic dinoflagellate with a well-known life-cycle, involv ing a vegetative thecate moti Ie stage, ecdysal stages and a benthic hypnozygote (cyst) [ I , 2]. This species has been commonly included in toxic algae lists, although its tox icity has been controvers ial for a long time. ince the late 1980' s th is spec ies has been associated with the occurrence of a new group of shellfish neurotoxins, yessotox ins [3, 41. L. polyedrum is known to be a common species along the Port uguese coast, although never associated with toxicity problems [5, 6]. In 1944, 50km north of Lisbon, this species was for the first time associated with a "redtide", involvi ng redd ish to brown water discoloratio n and intense night bioluminescence (7]. This was the first and the on ly published reference of a L. po/yedrum "red-tide" in PortugaJ, prior to the here reported e vent in J996. The present report includes data fro m the Portuguese HAll monitoring programme, an oceanographic cruise carried out in the affected area and data from a long-term dinoflagellate cyst study, started in 1996. MATERlAL AND METHODS Samples for the HAB monitoring programme were
Harmful Algal Dloorns 2000 HallegraefT, O.M., Blackburn, S.l., Bolch, C.J . and Lewis, R.I. (cds) Intergovernmental Oceanographic Commission of UNESCO 2001
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Atlantic Ocean Longitude Fig.l. Map of stud y area showing location of monitoring stations C), cross-shelf section (e) and cyst station (q». collected at different sites in Setubal Bay, by local fishermen (F ig.I). Sampling dates were dependent on shellfish colIecting activities in the area, and during the study period sam pling was intensified due to observation of water discoloration. At each site, 200m l of surface water samples were collected over natural shellfish beds (water column depth ca 30m). Samples were preserved upon collection with ac id ified formalin to a fi nal concentration of 1.2% . Phytoplankton counts were perform d using a PalmerMaloney chamber, after water concentration by centr ifugat ion (3000rpm for 20m in) and the lower limit of detection was of 100 cells.rI Results are expressed in celIs.r l. On I October, on board the RV "Capric6mio", a combined CTD/phyto plankton cross-shelf section was carried out in the Bay of Setubal (Fig.!). cm data were collected using a SBEI9 SEACAT pr filer. Wat r sam ples were collected with Nansen bottles at 5, 20 and 40m depth. Sampling was performed between 15 :00 and 17:00h . Samples were immediately preserved with hexamet hylenetetram ine buffered formalin to a final con entrat ion of 2.4 %. Phytoplankton cells were identified and counted by the UtermOhl method using a Zeiss IM35 inverted microscope with a detection limit of 20 cells.r l. L. po /yedrum ident ification was confirmed with SEM. For cyst studies, surface sediments (approximately the upper 1-2cm) were collected from a small recreational harbour, built in 1994 , inside the main port of Si.nes (F ig. I) . This site is located south of Serubal bay, and has
134 1000 - I
been used, since February 1996 and up to the present, as a long-term study site . Sed imen t sam ples were collected using a simp le sucking dev ice, and were kept cool and in the dark unt il treat ment. To assure no cyst germination occu rred . samples were processed the same day, or the follo w ing day . Sample pre paration involved sonication in an ultra-sonic bath , wet sieving through 150)J.m onto a 2S)J.m s ieve. and fu rther concentrat ion of this latter fr acti on us ing a hi gh density sol ut ion of sodium metatungstate (2.0 16g. cm· \ The lighter o rgan ic fraction, fl oated off by the heavy liquid, was then washed in dist illed w ate r, an d ali qu ots were mounted on m icroscopic slides with glycerine jelly [8 ]. Samples were exa m in ed for both e mpty cysts and cysts with cell content. Results are expressed as percentage values of the whole cyst assem blage. A total of at least 200 cysts were counted. Upwelling indices, based on the north-south wind compone nt, were calculated according to [9]. W ind data were obtai ned fro m the meteorolog ica l weather station located at the ca pe of S in es (In st ituto Nacional de Meteorologia, Boleti m Meteorol6gico Oiario).
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Fig. 3. Upwelling indices at the meteorological weather station of Sines. Negative values indicate upwelling and the black bars the days
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Fig. 2. Maximum concentrations of L. polyedrum in the area, at di ffe rent times.
Lingulodinium polyedrum was detected for the first time in SetUbal Bay on I I September, at levels of 4x 1oj cells.r I. Beca use the mo nitori ng program in the area involves samp ling of different stations at different dates, results show n in Fig. 2 refer to the maximum concentration values observed in the area at each date. On 19 Septem ber, the species already reached 71 Ox I oj cells.r and, on the 26th, similar high values were still recorded. Simultaneously, fishermen detected an intense brownish discoloration of seawater. On I October, data obtained during the oceanographic cruise at the surface of the innermost station suggested a marked decrease on the concentratio n of the species. On 4 and 19 October the monito ri ng program confi rmed the bloom decline. In 1996, th is was the last detection of L. polyedrum in the area. Wind data at S ines cape revealed that the Portuguese S W coast was under conditions of coastal upwelling during the sam pling period (Fig. 3). However, CTO data from the cruise carried o ut on I October, revealed that the water colu mn was well stratified , wi th an approximate g rad ient of 2.SoC between 10 and 30m depth, and with no
15
I
Distance offshore (km) ,
Fig. 4. Vertical distribution of (a) temperature (0C) and (b) salinity at the cross shelf section, I October. sign ificant vertical grad ient in salin ity (Figs. 4a-b). Phytoplankton data showed that L. polyedrum was present mainly in surface samples and above the thermocline (Fig. Sa). A maximum of 3xl0 5 cells.I· 1 was found ca. S km from the coast, at Sm depth, where the recorded temperature was 17.SoC and salinity was 36 (Figs. 4a-b). Cysts of L. polyedrum were also observed in the water column although concentrations never exceeded 80 cysts.!' I. Cyst distribution showed two maxima, one coinciding with the maximum of the vegetative stage when they represented 0.02% of the L. polyedrum population, and the other located near bottom level of the innermost station (Fig. 5b). The semi-quantitative cyst data on L. polyedrum, indicates that this species never reaches high values at Sines harbour. The highest values were recorded between October 1996 and February 1997, with a maximum of 12% recorded in January (Fig.6). It is interesting to note that in October and November 1996, the period immediately following the reported plankton bloom, cysts of L. polyedrum showed a marked increase in the cyst assemblage and all the recorded cysts had cell
135
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Fig. 7. Light micrographs of L. polyedrum: (a) vegetative ce lls (44-45 11m) and gamete (33 ~ m ) ; (b) ecdysal stage (44 ~m) ' (c) hypnozygote (47).Lm).
Cysts 20
15
10
0
Distance offshore (Ian)
Fig. 5. Vertical destri bution of (a) vegetative cells and (b) cysts of Lingulodinium polyedrum (cells. I-I) at the cross shelf section, I October. contents. Results also suggest that the annual maximum for this spec ies tends to occur in January . During the peak of the bloom two mai n size ranges of L. polyedrum cells were observed in the mo nitoring samples (Fig. 7a). The most abun dant ce lls were the larger ones and measured around 44-4 5 ~ m in length . The other group of cells measured approximate ly 331lm and appeared lighter in co lour. Ecdy sal stages were also observed (Fig. 7b). On Septemb r 26, not on ly these three types of cells were recorded but a lso fu lly devel oped resting cysts (diameter ca. 47~m) represe nti ng 0.1 % of the total recorded cells of L. polyedrum (Fig. 7c). During the bloom period, routi ne ana lysis (mouse bioassay and/or HPLC) of ASP, PSP and DSP biotoxins, carried out in the bivalves from .the area (Ens is siliqua and Callista chione), were all negative [ 10]. " 0' - - - - - - - - - - - - - -- - - -- - - - - - - - - - - - - - - - - ---,
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FMAMJJASO~DJ~MA~IJJA SONDJ ~MAMJJASONO JFMA MJJASO ~
1996
1997
1998
1999
Fig.6. Lingulodinium polyedrum cysts at Sines harbour DISCUSSION
L. polyedrum is a cosmopoli tan , euryhali ne, wann temperate species, with the no rthern di stri bution lim it of its cyst linked to the sub-polar/temperate cyst boundary in the northern North Atlantic [2, II] . he review article on L. polyedrum reports different conditions of stratification and nutrient enrichment, in diffe rent geographical areas, leading to bloom development of the vegetative stage [2] . These observations support th e present day eco logical interpretation of the increase in the cyst assemb lage of L. polyedrum as a nutrient enrichm ent signal, namely
eutrophication or upwelling, rather than a low sali nity signal as previously suggested [ I I]. Here we report a bloom of L. polyedrum observed in late summer-early autumn , when the Portuguese coast is characterised by upwelli ng. On the west coast of Portugal this phenomenon is mainly d pendent on the occurrence of no rtherly winds . Dur ing the sampling period , the upwelling indices at cape Sines suggested that th is process was occurri ng a long t he southwest coast. However,. the r suits of the cross-shelf ocean ograp hi c transect ind icated that the water column inside Setubal Bay, where the b loom was recorded . was cl ear ly stratified . This may be expla in ed by several reg ional features (Fig I) : (i) the bay is lim ited north by the mo untain range Serra da Arrab ida , suggesting th is formation may act as a barr ier to the local infl uence of northerly winds ; (ii ) waters upwe lled north of cape Espichel are advected southwards , althou gh flowing o ffshore the samp led area, d ue to a pronou nced coast di scontinuity at cape Es pichel ; (i ii) the gen e ral topography of the area [ 12]. This author showed sev ral sate ll ite images confi rm ing the presence of warmer waters inside the Bay bordered by cold upwelled waters. During the cruise, the spec ies max imum was recorded at the surface during th mid-afternoon. Th is observati on is in agreement with the described vertical diel-migration behav iour of L. p olyedrum [2, 13] . Prev ious reports on the appearance of L. p olyedrum cysts in the water c lu mn, as described for other dinoflage llates, show that these tend to occur after an in crease in population growth, towards the end of a bloom [I , 14]. On 26 September and during the m ise ( I October), cysts with ce ll con tent we re already observed in the water co lumn , suggesting L. polyedrum had reached its maxi mum, and the bloom was already decl ining inside the bay. Along the cross-shelf section, the maxi mum of cysts observed near the su rface suggests encystment was occu rring, while the maximum near-bottom , below the th ermoc line, may be associated wi th encystment and sinking that took place in the previous days (Fig. 5b).
136
During this study and on 26 September, the maximum ratio of cysts to vegetative cells was I: 1000. This ratio is significantly lower than the ratios observed for other closely related species, such as Gonyaulax grindleyi (I :2) or Gonyaulax digilale ( I :500) [15]. Reports from a loch on the West Coast of Scotland give a time-lag of less th an one month between the presence of cysts in the water column and the increase in . newly formed cysts in the sediment record [ 14]. Our data aJso suggests the same pattern. Cysts of L. polyedrum in sed iments collected during October and November 1996 were only recorded with viable cell content. In January 1997, although empty cysts showed an increase, cysts with cell content reached a ma:omum . Since 1996, the monitoring program has not detected blooms of L. polyedrum. However, the cyst assemblage shows, for the following years, what may be considered a seasonal pattern, with maximum values occurring in January (Fig. 6). These results suggest that in the southwest coast of Portugal this species may have a seasonal cycle with a general trend simi lar to what has been described in higher latitudes [2]. During the peak of the bloom, the different size and colour of the swimming cells match the description from the I iterature for vegetative cells and gametes [16]. These, together with the presence of resting cysts and ecdysal stages suggest that almost all the life cycle stages of L. polyedrum were present simultaneous ly. Sexual reproduction was apparently occurring as evidenced by the presence of gametes and hypnozygotes (Fig. 7). In 1993, it was suggested for the first time that ecdysal stages could be transformed directly into resting cysts [17] . However, the author referred those conclusions could be speculative since the original motile population used in the experiments could have already undergone sexuality. Our field observations conftrm that the different life cycle stages can o-occur and sexuality may be coinciden t with the presence of ecdysal stages. To our knowledge, his is the first fi eld report that clearly identifies the simultaneous occurrence of a\l these li fe stages in the water column. Although nutrients have not been measured, we consider that the influence of upwelled waters, offshore the bay, is a likely source of nutrients for these stratified waters. as observed to occur between upwelling events in the system of southern California [18]. It is therefore iot resting to note that cyst assemblages influenced by upwelling off California are dominated by L. polyedrum cysts [11]. Our results support the hypothesis that stratification and nutrient enrichment are th e main requireme nts for the development of a L. p olyedrum bloom [2]. However, the origin of stratification (thennal or haline) and the source of nutrients (e.g. anthropogenic, upwelling) seem to be irrelevant. Although L. polyedrum has been associated with yessotoxins production and shellfish toxicity [3, 4], the results of biotoxin routine analysis dur ing the bloom period continn that no harmful effects are associated with L. po/y edrum in the Portuguese coast. From a regional point of view, it is also interesting to no Ie that winds measured at the meteorological station of Sines can not be considered as indicato rs of
stratification/mixture cycles in the Bay of Setubal, and consequently can not be used as too ls to predict blooms in the bay.
ACKNOWLEDGEMENTS This work was partly funded by project EUFlTOX, PRAXIS XX], 2/2. 1IMarI1 736/95. We thank Dr. Tere a Rosa for provid ing physical data
REFERENCES I. D. Wall and B. Dale, Micropaleontology, ll: 265-304 (1968). 2. J. Lewis and R. Hallet, in: A.D. Ansel~ R.N . Gibson & M.Bames (eds.), Oceanography and Marine Biology: an Annual Review, UCL Press, J1, pp. 97-1 61 (1997). 3 . M. Kumagai, T. Yanagi. M. Mumta, T. Yasumoto, M. Kat, P. Lassus and l.A . Rodriguez-Vasquez, Agriculture Biology Chern istry iQ.: 2853·2857 (1986). 4 . A. Tubaro. L. Sidari, D. Loggia and T. ¥ asumoto, in: B Reguera, 1. Blanco, M.L. Fernandez & T. Wyatt (eds.), Harmful Algae, Xunta de Galicia and IOC of UNESCO, pp. 470-472 (1998). 5. E.S. Silva, Scparatas dos Arquivos do Instituto Nacional de SaiIde (Lisboa), IY.: 252-262 (1980). 6. M.T. Moita and M.G. Vilarinllo. Portugalia Acta Biol6gica (Lisboa), ser.B, Sist, il.: 5-50 (1999). 7. 1.S. Pinto. Boletim da Sociedade Portugucsa de iencias Naturais (Lisboa), fl. Ser.2 rom: 94-97 (1949). 8 . A. Amorim and B. Dale, in: B. Rcguera, J. Blanco. M.L. Fernandez & T. Wyatt (eds.) Harmful Algae. Xunta de Galicia and JOe of UNESCO, pp. 64-65 (\998). 9. A. Bakun. NOAA Tech. Rept. NMFS SSRF-67J. I03pp ( 1973). 10. MA Sampayo, S. Franca, I. Sousa, P. Alvito, P. Vale, M.1. Botelho, S. Rodrigues and A. Vieira. Arquivos do Instituto Nacional de SaUde (Lisboa), 23: 181-188 (1997). II. B. Dalc, in: 1. Jansonius & D.C. MacGregor (cds.). Palynology: principles and applications, AASP Foundation, 1. pp. 1249·1275 (1996). 12. A. riUza., in : E. Suess & l . Thiede (eds. ) Coastal Upwelling Its Sediment Record -Part A: Response of the Sedimentary Regime lo present Coastal upwelling, Plenum Press. New York. pp. 85-98 (1983). 13. D. Blasco, in: V.R. loC icero (ed.). Tox ic Dinofagella.J.e blooms., Wakefield MA : Massachusetts S i. and Tech .. Found., pp. 11 3- I 19 (1975). 14. I. Lewis P. Ten IUld 1. 0 . Dodge, in: D.M. Anderson, A.W. White & D.G. Baden (eds.). Toxic dinoflagellates. Elsevier, New York. pp. 85·90 (1985). 15. B. Dale, Review of Palaeobotany IUld Palynology, 22: 39-60 (1976). 16. J.P. Kokinos and D.M Anderson, Palynology, 12: 143·166 ( [995). 17. L Marasovic., in: T.J. Smayda & Y. Shimizu (eds.) T OXIC Phytoplankton Blooms in the Sea, Elsevier, Amsterdam. pp. \)9-1 42 (1 993). 18. R.W. Eppley and W.G. Harrisson, in: V.R. loCicero (ed.), Toxic Dinofagellate blooms, Wakefield MA: MassachuSCI15 Sci. and Teen.. Found., pp. 11·22 (1975).
