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Survival and photosynthetic activity of different Dinophysis acuminata populations in the northern Baltic Sea
Harmful Algae 4 (2005) 337–350 www.elsevier.com/locate/hal
Survival and photosynthetic activity of different Dinophysis acuminata populations in the northern Baltic Sea Outi Seta¨la¨a,b,c,*, Riitta Autiob, Harri Kuosac, Janne Rintalab,c, Pasi Ylo¨stalob a
Finnish Environment Institute, Mechelininkatu 34a, P.O. Box 140, FI-00251 Helsinki, Finland b Finnish Institute of Marine Research, P.O. Box 33, FI-00931 Helsinki, Finland c Tva¨rminne Zoological Station FI-10900 Hanko, Finland Received 25 February 2004; accepted 14 June 2004
Abstract This study deals with a recently found phenomenon in the northern Baltic Sea: the occurrence of the dinoflagellate Dinophysis acuminata in the deep water below the thermocline. This was first observed in July 2001 at the station BY 15 in the Gotland Deep, where a sharp and intensive chlorophyll fluorescence signal was encountered at 77 m depth. The fluorescence peak was due to a dinoflagellate community dominated by Dinophysis acuminata (approximately 18 000 cells l1). The survival of this community was followed in laboratory incubations in low light (20 mE m2 s1) and low temperature (+5 8C). After 5 weeks incubation, 67–84% of the initial cell abundance was lost, while few D. acuminata cells survived up to 24 weeks in the original sample. During the incubation, the fluorescence signal of the cells became fainter and the chloroplasts smaller and aggregated. On two occasions a D. acuminata cell was found attached to a smaller cell by a thin cytoplasm strand, possibly indicating mixotrophic behavior. During the following summer (2002), the photosynthetic efficiency of D. acuminata collected from thermocline layers of few stations and from the nitracline (75–80 m) at one station was studied in photosynthesis irradiance (P–E) incubations. Photosynthetic activity occurred in all populations, with differences in their photosynthetic carbon uptake rates. Photosynthesis of D. acuminata populations was saturated between 250 and 500 mE m2 s1; maximum cell-specific carbon uptake rates (Pm) ranged from 160–925 pg C cell1 h1. The Pm-rates in populations originating below the thermocline and in an artificially darkened population were markedly lower than in populations from upper water layers. The varying maximum photosynthetic rates of these populations may reflect their history, e.g. time spent in different light environments. # 2004 Elsevier B.V. All rights reserved. Keywords: Survival; Carbon uptake; Vertical migration
1. Introduction * Corresponding author. Tel.: +358 9 40300292; fax: +358 9 40300291. E-mail address: [email protected] (O. Seta¨la¨).
Dinophysis acuminata and Dinophysis norvegica are common and frequently abundant dinoflagellate species in the northern Baltic Sea. These species often
1568-9883/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2004.06.017
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dominate the late summer algal blooms in the Gulf of Finland (Kononen and Niemi, 1986; MeyerHarms and Pollehne, 1998; Kononen et al., 1999, 2003; Gisselson et al., 2002). In the brackish central and northern Baltic, they occur usually at concentrations of 102–103 cells l1 (Carpenter et al., 1995; Gisselson et al., 2002; Salomon et al., 2003), and occasionally reach higher concentrations of up to 150 103 cells l1 (Carpenter et al., 1995). When abundant in late summer and autumn, autotrophic Dinophysis spp. are regularly found concentrated at the thermocline in 15–25 m (Niemi, 1975; Kuosa, 1990; Carpenter et al., 1995; Gisselson et al., 2002). The relationship between Dinophysis blooms and the environmental factors has been studied by several authors (e.g. Delmas et al., 1992; Subba Rao et al., 1993; Carpenter et al., 1995; Figueroa et al., 1998; Gisselson et al., 2002; Godhe et al., 2002). The conditions leading to Dinophysis spp. blooms are of special interest, since many Dinophysis species produce toxins that may cause severe economical problems in mussel farming areas (Hallegraeff, 1995). In the Baltic Sea okadaic acid, a toxin produced by Dinophysis species, has been observed in the soft tissues of blue mussel (Pimia¨ et al., 1998) and in flounder liver (Sipia¨ et al., 2000). Many studies have shown the connection between high abundances of Dinophysis spp. and stratification (e.g. Lassus et al., 1988, 1991; Delmas et al., 1992; Reguera et al., 1995). On the other hand, no evidence of blooms promoted by elevated nutrient concentrations has been found. Instead, both thermal (Delmas et al., 1992) and salinity induced stratifications (Giacobbe et al., 1995; Peperzak et al., 1996; Godhe et al., 2002) have been associated with high Dinophysis abundances. Dense blooms of Dinophysis spp. along the French Atlantic coast were observed both in nutrient-poor and nutrient-rich waters, and the main factor promoting Dinophysis spp. growth was concluded to be thermal stratification that lasted for over 2 weeks (Delmas et al., 1992). In Bedford Basin, eastern Canada, D. norvegica bloomed when a strong density gradient suppressed turbulence allowing the cells to concentrate (Subba Rao et al., 1993). Neither study found that Dinophysis blooms would depend on the availability of dissolved nutrients.
Autotrophic dinoflagellates perform diel vertical migration (DVM) in the water column that is typically induced by taxic responses to light and/or gravity. Changes in the biochemistry over the cell cycle (Kamykowski, 1995), cellular nutrient ratios (Kononen et al., 2003) and chemical and thermal gradients (Cullen and Horrigan, 1981; Cullen, 1985; MacIntyre et al., 1997) influence the distribution and migration patterns of dinoflagellates. The DVM patterns express near-surface aggregations during daylight with descent or dispersal during the night. In the Baltic Sea, the Dinophysis spp. occurring at relatively low light environments at the thermocline have not been observed to perform vertical migrations. This has raised questions about their ability to provide carbon for their growth by photosynthesis in environments where light supply is limited (Carpenter et al., 1995; Gisselson et al., 2002). In Ria de Vigo, NW Spain, Dinophysis acuminata has, however, been found to migrate vertically between surface and 10 m depth (Villarino et al., 1995). In the same area, Figueroa et al. (1998) found D. acuminata cells aggregated close to the surface at noon and descended during the night. In this shallow stratified estuary, the cells, however, were seemingly unable to swim through a well-defined pycnocline. Attempts to culture Dinophysis spp. in various culture media, or with accompanying phytoplankton species, have shown an ability of the cells to survive on their own cellular reserves for a few weeks, and, at best, to grow and persist for 16–20 weeks (e.g. Nishitani et al., 2003; Sampayo, 1993). Generally the cells become thinner and less pigmented with time, with reduced and deformed plasmatic contents (Sampayo, 1993; Maestrini et al., 1995, this study). So far, all attempts to establish a true culture of Dinophysis spp. in media that support the growth of other phytoplankton species have failed (Keller and Guillard, 1985; Sampayo, 1993; Maestrini et al., 1995; Subba Rao, 1995; Grane´ li et al., 1997; Nishitani et al., 2003). A routine CTD-cast in the Baltic Proper revealed a sharp chlorophyll fluorescence signal at 77 m. The water was found to contain a dense community (approximately 18 103 cells l1) of Dinophysis spp. at a depth where no light to support photosynthesis is available. Preliminary studies (laboratory incubations) were conducted to follow the survival capacity
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of the D. acuminata cells in this deep-water community. During the following summer (August 2002) the studies were continued by evaluating the photosynthetic activity of cells collected below the thermo- and halocline from open sea, deep-water stations and from the thermocline of one coastal sampling site.
2. Material and methods 2.1. Sampling Most of the material for this study was collected in the Gotland Sea onboard R/V Aranda (Finnish Institute of Marine Research) during two research cruises in August 2001 and 2002. The data set for year 2001 includes the first observation of a deep-water community of Dinophysis spp. from a Gotland Sea station (BY 15, the Gotland Deep), and the laboratory incubations. The data set from August 2002 includes the P–E measurements of different Dinophysis acuminata populations collected from an open-ocean station in the northern Baltic Sea (LL 23; total depth 459 m) and from a coastal station (Tva¨ rminne Storfja¨ rd, total depth 33 m). Temperature, salinity and the in situ Chl-a fluorescence were measured with a SBE 911 Plus CTD-probe (SeaBird Electronics) equipped with a SeaTech (440/680) fluorescence sensor. Dissolved inorganic nutrients, oxygen, H2S and Chl-a concentrations were analyzed according to standard methods (Grasshoff et al., 1983). Temperature, salinity and nutrient profiles from Tva¨ rminne Storfja¨ rd are presented for the days of measurement closest to the actual sampling dates of D. acuminata populations. Samples for phytoplankton counts were preserved with acid Lugol’s solution (Ha¨ llfors et al., 1979), and settled in 50 ml tubes for 24 h following Utermo¨ hl (1958), and counted with inverted microscope (Leica DMIL, 100–400 magnifications). Samples were also fixed with 25% glutaraldehyde (final conc. 1–2%) and studied with Leica DMIL inverted microscope with epifluorescence setup under green (546/14 nm, 580 nm beam splitter, 590 nm barrier filter) and blue excitation light (450–490 nm, 510 nm beam splitter, 515 nm barrier filter), in order to study the condition and distribution of chloroplasts of the cells.
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2.2. The survival of Dinophysis acuminata in laboratory incubation As a pilot study, the viability of the cells was studied in laboratory incubations. Two 8 l polycarbonate bottles were filled with water collected from 77 m at the Gotland Deep station (year 2001), and kept in an incubation room (+3 8C, darkened) onboard R/V Aranda for 7 days (until the end of the cruise). Further incubations were made in an onshore laboratory in four 4 l polycarbonate bottles, each containing 3.5 l of water and kept at +5 8C exposed to a 12:12 h light:dark cycle under 20 mE m2 s1 with minimal mechanical disturbance. Since the concentrations of the inorganic nutrients at the start of the incubation were high (NH4–N: 1.2 mmol l1, NO3 + NO2–N: 9.4 mmol l1, PO4–P: 2.8 mmol l1), nutrient additions were not made. Because D. acuminata occurred in darkness, mixotrophy was speculated to play a role in their nutrition. Live, cultured microalgae and the ciliate Mesodinium pulex were added to the incubated samples (=units) at low concentrations (algae: 250– 400 cells ml1, M. pulex: 5 cells ml1) to see whether D. acuminata would benefit from the presence of other protists. The accompanying species were: Heterocapsa triquetra and Heterocapsa rotundatum (unit B = dinoflagellate unit), Prorocentrum minimum, Storeatula major and Mesodinium pulex (unit C = mixed unit), Storeatula major and Pyrenomonas sp. (unit D = cryptophyte unit). Unit A was the untreated control. The added algae had been grown under 12:12 h light:dark cycle (40–60 mE m2 s1) at 18 8C in f/2 (– Si) medium (Guillard, 1975). The ciliate Mesodinium pulex had been cultured in a seawater medium containing 0.1 ml l1 of f/2 iron-EDTA trace metal solution (Guillard, 1975) with Storeatula major as prey. The survival of D. acuminata cells in each treatment was followed weekly by examining live samples under stereomicroscope (6.3–50 magnification, Leica MZ 7.5) to assess their condition, and to obtain a rough estimate of their abundance. This was done by carefully pouring water into a petri dish and letting it settle for approximately 15 min, after which swimming D. acuminata cells were easily detected. Samples for more detailed microscopy examinations were fixed with glutaraldehyde or with acid Lugol’s solution (Ha¨ llfors et al., 1979). Regular quantitative
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sampling was not considered, since it would have consumed too much of the experimental water; therefore, the outcome of these incubations was solely observational. 2.3. P–E measurements Photosynthetic carbon uptake rates (P–E measurements) of different D. acuminata populations were measured in August 2002. Since the cells were dispersed in the water column in abundances of 1–2 103 cells l1, the water was concentrated following Maestrini et al. (1995). The water sample was successively poured through two different mesh sizes (100 and 20 mm) and the filtrate gently siphoned. If the filtrate contained a marked number of cells other than Dinophysis, it was further filtered through a 30–50 mm net, using an inverse method in which water is carefully sucked through the filter with a syringe. With this procedure, the D. acuminata cells were left in the water and smaller cells excluded. If the ciliate Mesodinium rubrum was abundant in the sample, which often was the case, the water was gravity filtered through a 63 mm net, a method that eliminates the fragile M. rubrum cells which explode during filtration, but allows most of the D. acuminata cells to pass through undamaged. The filtrate was settled for 2–24 h, after which the upper part of the water was gently poured into a 1–2 l polycarbonate bottle. This allowed only actively swimming cells to be incubated. A 5–10 ml sample from the filtrate was fixed with Lugol’s solution, pipetted into Utermo¨ hl chambers, and the number of cells in the sample counted. This filtration protocol yielded concentrations of 30–160 D. acuminata cells ml1 for the P–E measurements. The cells from the open-sea station, LL 23, were sampled at the thermocline at 14 m and from deep water at 80 m. Two sets of samples were collected at the coastal station, Tva¨ rminne Storfja¨ rd, from the thermocline at 15–17 m and from 20–22 m (2 and 7 August). All P–E incubations were done on the sampling day, except the other half of the cells from Tva¨ rminne Storfja¨ rd sampled on 2 August, that were incubated for 6 days in darkness (in situ temperature) before measurement. Photosynthetic carbon uptake was measured as 14 C-CO3 incorporation in a P–E incubator with 16 different light levels, and 2 dark incubations serving as
controls. The light levels were provided with a 300 W quartz halogen lamp covered with heat shields and with an output of 2475 mE m2 s1 and measured with a scalar irradiance meter (Biospherical Instruments, QSL-2101). Duplicate sets of the D. acuminata concentrates (volume 3 ml) were incubated for 2 h in 7 ml glass scintillation vials. One microcurie of NaH14CO3 (DHI Water & Environment, Denmark) was added to the samples prior to the incubation, which were terminated by adding 100 ml of 38% formaldehyde to the samples. Net primary productivity was measured by removing the remaining inorganic carbon by adding 100 ml 1N HCl to the vials and letting them stand open for 48 h. After the acidification, 4 ml of scintillation cocktail (Instagel+, Packard, USA) was added to the vials and the radioactivity measured with a scintillation counter (LKB Wallac RackBeta, Finland). The average DPM count value of duplicate dark bottles was subtracted from each sample. Cell-specific carbon uptake was calculated as P ðDPMcell DPMdark Þð CO2 ÞVk1 P¼ DPMadded T where P is the cell-specific carbon uptake, DPMcell the DPM count from a sample divided by the number of D. acuminata cells in that sample, P DPMdark the DPM/cell from the dark controls, CO2 the total dissolved inorganic carbon in the water, V the sample volume (3 ml), k1 the correction factor for isotope discrimination (1.05, Æertberg-Nielsen and Bresta, 1984), DPMadded the DPM added to each vial, and T the incubation timePin hours. In brackish waters, such as the Baltic Sea, CO2 can be calculated according to the formulas of Buch (1945). The P–E parameters for the comparison of different curves were calculated using a modified version of the exponential model according to Platt et al. (1980): B B a E b E B B P ¼ Ps 1 exp exp PB PB s s where PB [pg C cell1 h1] is the cell-specific rate of carbon incorporation at irradiance E, aB [(pg C cell1 h1) (mE m2 s1)1] the initial slope of the P–E curve, E the irradiance [mE m2 s1] and bB [pg C (cell1 h1) (mE m2 s1)1] the photoinhibition parameter, PB s the equivalent to the maximum
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rate of cell-specific rate of carbon incorporation, PB m [pg C cell1 h1], when bB = 0. Otherwise when bB 6¼ 0, realized cell-specific maximum rate of carbon incorporation, PB m is calculated according to Platt et al. (1980) as follows: PB m
¼
PB s
aB a B þ bB
bB a B þ bB
b=a
The light saturation parameter, Ek [mE m2 s1] is B calculated as Ek = PB m /a . 3. Results 3.1. Hydrography, inorganic nutrients, Chl-a and plankton community In 2001, a well-defined thermocline was situated at 13–17 m depth at station BY 15. The deeper water column was separated by a halocline at 60–80 m (Fig. 1). The oxygen concentration declined rapidly below 70 m, and traces of H2S were detected at 125 m and below. The concentrations of inorganic nutrients in the water column above thermocline were low (PO4–P and NO3 + NO2–N: <0.05 mmol l1, NH4–N: <0.20 mmol l1), but increased markedly below 50 m. The highest concentration of inorganic nitrogen was measured at 80 m depth (NO3 + NO2–N: 9.4 mmol l1). Chl-a concentrations peaked at 10 m depth (2.23 mg Chl-a l1) and again at 77 m (0.92 mg Chl-a l1). The measured hydrographical parameters and inorganic nutrient concentrations at station LL 23 in year 2002 were similar to those described for the Gotland Deep (BY 15) in the previous year, except for somewhat higher temperatures. The hydrography of the coastal station, Tva¨ rminne Storfja¨ rd, reflected warm and calm weather conditions. The salinity was approximately 5 psu through the whole water column, and temperature was >20 8C down to the thermocline at 15–17 m. The inorganic nutrient concentrations increased with depth. In year 2001, the phytoplankton community in the euphotic layer consisted mainly of small, <10 mm flagellates. Filamentous blue-green algae were practically absent. Dinoflagellates formed 2.8–9.8% of the total autotrophic phytoplankton biomass in 0–12 m.
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Dinophysis spp. were absent, or in low abundance at all depths except at 77 m, where 18 103 cells l1 (37.8 mg C l1) were found. Approximately 95% of the cells were identified as Dinophysis acuminata, and the rest were D. norvegica (M. Huttunen, Finnish Institute of Marine Research, (pers. comm.). The phototrophic ciliate Mesodinium rubrum was abundant in the upper water column (2.4–6.1) 103 cells l1). The phytopankton community in the euphotic zone in the year 2002 sample consisted mostly of large colonial or filamentous blue-green algae, (<3 mm) and auto- and heterotrophic flagellates. The proportion of blue-green algae above the thermocline increased with depth. As in 2001, the proportion of dinoflagellates, including Dinophysis spp., was insignificant in the 0–12 m water layer. Dinophysis acuminata was found in low concentrations (<2 103 cells l1) between 70 and 80 m, as was M. rubrum (<100 cells l1). The phytoplankton community in year 2002 in Tva¨ rminne Storfja¨ rd consisted of filamentous blue-green algae (mainly Aphanizomenon flos-aquae and small Pseudoanabena species) and small autotrophic flagellates (Chrysochromulina spp., Pseudopedinella spp., Cryptomonadales and <3 mm unidentified cells). The phytoplankton community at the thermocline region was similar, except for declining abundance of Aphanizomenon flos-aquae and the presence of Dinophysis spp. 3.2. Laboratory incubations The numbers of D. acuminata cells decreased in all experimental treatments during the first week. On the fifth laboratory incubation week (7 weeks after initial sampling from 77 m), 67–84% of the original cell abundance had been lost, and 3–6 cells ml1 in three of the four units were found. In experimental unit B the vigorous growth of Heterocapsa spp. during the first 2 weeks of incubation, and the successively decaying phytoplankton material either overgrew D. acuminata or masked it, since living D. acuminata cells were not found in this unit. Motile D. acuminata cells were abundant on week 7 in units A (control) and D (cryptophyte), whereas only few cells were routinely found in the mixed population unit C. Between weeks 7 and 16, swimming cells were found in units A and D, but only occasionally in unit C. On
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Fig. 1. (A–I) Profiles of temperature, T (8C), salinity, S (psu), oxygen, O2 (ml l1), nutrient concentration (mmol l1), Chl-a (mg l1), CTD fluorescence (arbitrary units) and D. acuminata biomass (mg C l1) at the sampling stations in years 2001 and 2002.
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week 16 of the experiment incubation of the units C and D was terminated since no living D. acuminata cells were observed. In February 2002, 6 months after the initial sampling, few slowly swimming D. acuminata cells were still found in the control unit. Dividing cells were not observed in any treatment during the incubations.
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distributed and tightly packed, as described above, and more or less rounded vacuole-like bodies were observed. These bodies were of different sizes and transparent under transmitted light. Occasionally, a whole cell was green under blue excitation light (Fig. 3D). On two occasions a Dinophysis acuminata cell was found attached to a small (approximately 7– 8 mm) cell by a thin cytoplasm strand (Fig. 2E and F).
3.3. Observations on Dinophysis acuminata cell structure
3.4. Photosynthesis
In the beginning of the incubation period, the cells were mostly in good condition, with branched, brightly fluorescent yellowish-orange chloroplasts. Thereafter the chloroplasts of the incubated cells generally became smaller and densely packed (Fig. 3A and B), the fluorescence signal became fainter, and finally (after 6 months incubation) only one or two ‘‘packages’’ of faintly coloured chloroplasts in otherwise transparent cells were seen (Fig. 3C and D). Various types of ‘‘deformations’’ in the cell contents were seen with epifluorescence microscopy from week 5 onward. The chloroplasts were unevenly
All D. acuminata populations were photosynthetically active. The P–E parameters and the corresponding P–E curves from the incubations for each population are presented in Table 1 and Fig. 2. The populations could be divided into two types based of their specific P–E characteristics and are referred to as the Type I and Type II populations. Type I includes photosynthetically ‘‘very active’’ populations (Tva¨ rminne Storfja¨ rd: 15–17 m and LL 23: 14 m), with light-saturated maximum photosynthetic carbon uptake (Pm) of 782 and 925 pg C cell1 h1. The a values describing the light limited initial slope were
Fig. 2. (A–E) P–E curves for different D. acuminata populations in year 2002. (A–C) Tva¨ rminne Storfja¨ rd (Tv): A = thermocline, B = the population in A after 6 days dark incubation, C = below thermocline. (D, E) Landsort deep (LL 23): D = thermocline region, E = deep-water layer. For the curve fitting see Section 2.
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Table 1 P–E parameters of the Dinophysis acuminata populations measured in August 2002 Station
a b Pm Ek
Tv 2.8. (depth = 15–17 m)
Tv 2.8. (depth = 15–17 m, darkened)
Tv 7.8. (depth = 20–22 m)
LL 23 14.8. (depth = 14 m)
LL 23 14.8. (depth = 80 m)
6.29 0.22 783 124
1.90 0.56 361 190
2.11 0.19 210 100
9.74 1.12 925 95
2.19 0.06 160 72
Tv = Tva¨ rminne Storfja¨ rd, LL 23 = Landsort Deep. Description of the parameters: a = light-limited initial slope [(pg C cell1 h1) (mE m2 s1)1], b = parameter describing the reduction in photosynthetic rate with high irradiance [pg C (cell1 h1) (mE m2 s1)1], Pm = light-saturated maximum rate of photosynthesis (pg C cell1 h1), Ek = light saturation parameter (mE m2 s1).
high (6.29 and 9.74) in these two populations collected closest to the surface, indicating a rapid increase in the production rate with increasing irradiance. The other three populations (Type II populations) were characterized by low/lowered photosynthetic activity, expressed as low Pm (160–361 pg C cell1 h1) and a (1.90–2.19) values. The two measurements made with the populations from below the thermocline (Tva¨ rminne Strorfja¨ rd: 20–22 m and LL 23: 80 m) had the lowest Pm values (Fig. 2C and E). The population collected from Tva¨ rminne Storfja¨ rd from 15–17 m depth and kept in darkness for 6 days prior to the P–E incubations had somewhat higher measured Pm levels than the deep-water samples (Table 1, Fig. 2B). In all populations, photosynthesis was saturated at irradiances between ca. 250 and 500 mE m2 s1. The b value describing photoinhibition was highest in the 14 m population from open-sea station LL 23. The dark uptake rates of 14C were <10% of the maximum uptake rates in all incubations.
4. Discussion The Baltic Sea is characterized by a high concentration of coloured dissolved organic matter that affects the attenuation of solar irradiance. As a consequence of the overall high light attenuation in the Baltic Sea, the euphotic zone (>1% of surface light, PAR) does not exceed 20–30 m (e.g. Aarup, 2002). Because of these properties, the solar irradiance in the northern Baltic does not penetrate below the halocline, which also means that the Dinophysis spp. cells collected from 77 and 80 m evidently occurred in
darkness. The occurrence of these cells in such an extreme and isolated environment suggests that these species may migrate through strong gradients, and persist at conditions unsuitable for any active and primarily phototrophic organism. The laboratory incubations of this study showed that a part of the deep-water Dinophysis acuminata population was able to survive in rather extreme conditions for up to 6 months, although in the end only few active cells were found. Their survival indicates the ability of D. acuminata to persist for a relatively long time period in unfavorable conditions. This is of special interest in light of the difficulties met in earlier culturing attempts. Recently, Nishitani et al. (2003) tried to cultivate Dinophysis caudata together with accompanying phytoplankton (Thalassiosira sp., Chroomonas sp. and an eukaryotic picoplankton species) as possible prey. At best, they were able to grow 28 cells from one D. caudata cell, and keep them alive up to 2 months, which may indicate that D. caudata benefited from the addition of other protists. This, however, does not conform to our observations of D. acuminata survival in the different experimental units, where no clear benefit of the accompanying species was found. One reason for successful growth in the study by Nishitani et al. (2003) could have been that the algae were added crushed and frozen before addition, and thus did not compete with D. caudata cells. Our incubations revealed that the size and shape, and the fluorescence intensity of the chloroplasts diminished with time; the last observed living cells were almost transparent (Fig. 3C). Lucas and Vesk (1990) suggested that in recently divided cells the
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Fig. 3. (A–F) Various types of Dinophysis acuminata cells. (A, B) Cells with beady chloroplasts, (C, D) cells near termination of incubation with only few packages of chloroplasts, (E, F) a D. acuminata cell attached to a smaller cell. (A, C, E) Light microscopy, (B, D, F) epifluorescence microscopy.
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chloroplasts appear scattered through the cytoplasm, whereas in older cells they aggregate into clumps. The chloroplasts of the cells collected from the deep-water layers during both years appeared, however, to be mostly typical of those for healthy, and presumably recently divided cells (bright and branched). If the recorded changes in the size and shape of the chloroplasts were typical and related to their aging process, then the occurrence of the Dinophysis spp. cells in the deep-water samples must have been the result of some relatively recent process. Mixotrophy by phagotrophy or osmotrophy has been suggested to be an important factor supporting the nutrition of Dinophysis spp. (e.g. Jacobson and Andersen, 1994; Carpenter et al., 1995; Grane´ li et al., 1997). Jacobson and Andersen (1994) found food vacuoles inside D. acuminata and D. norvegica, which was first-hand evidence of their phagotrophic capability. Based on the food vacuole contents, these authors suggested that these autotrophic Dinophysis spp. were preying on ciliates in the same manner as the heterotrophic Dinophysis rotundata observed to feed on ciliates of their own size (Hansen, 1991). Nishitani et al. (2002) observed picoplanktonic algae attached to the surface of Dinophysis spp. and food vacuole-like structures filled with red fluorescence probably from algal prey. Although no clear relationships between the abundance of picoplankton and Dinophysis spp. were detected, Dinophysis spp. were suggested to feed on them. Signs of mixotrophy was also found by Ishimaru et al. (1988), who observed how attached cryptophytes were absorbed inside Dinophysis fortii. Our images of D. acuminata cells in contact with a small cell can be interpreted in several ways. Both Subba Rao (1995) and Berland et al. (1995) have reported the development of unknown, round bodies inside D. acuminata cells, and their release. Some of these bodies were packed with small granula, suspected to be zooids or gametes. Our images of the two connected cells resemble those illustrated by Subba Rao (1995). However, the vacuole-like bodies that we observed inside D. acuminata were of various size and shape, and we did not observe gametes or zooids. The occurrence of deformed, beady-looking chloroplasts in Dinophysis spp. has been noted by several authors. Noren et al. (1999) reported granular chloroplasts in D. norvegica cells infected by the parasite flagellate Parvilucifera infectans. Infection of
D. norvegica populations by another parasite, Amoebophrya sp. trophonts was reported by Gisselson et al. (2002) and Salomon et al. (2003). Our Dinophysis acuminata populations also showed signs of Amoebophrya infections, but the prevalence appeared low (approximately 5%; Wayne Coats, Smithsonian Environmental Research Center, pers. comm.). It remains unclear whether the connected Dinophysis acuminata cells were releasing gametes/zooids, under a parasite attack, or actually feeding on the smaller cells. Since the photosynthetic activity of the Dinophysis spp. community in year 2001 sample was not measured, the ‘‘condition’’ or viability of the cells was estimated by microscopical observations only. However, the P–E measurements made in the following year showed that the deep-water community consisted of active cells. Populations originating from different depths varied in their maximum photosynthetic carbon uptake rates (Pm values). The lowest Pm values were obtained from the populations collected below the thermocline, and were lower than the values of the artificially darkened population. This may reflect the history of these populations, i.e. the longer the time period spent in darkness, the lower the Pm value. In the two populations from the thermocline layer, the maximum photosynthetic carbon uptake rates were markedly higher (Pm: 783 and 925 pg C cell1 h1). Photosynthetic carbon uptake rates of Dinophysis norvegica were measured by Gisselson et al. (2002) using light levels of 8– 330 mE2 s1. Their measurements were done with one population collected from 18 m depth, and gave saturated carbon uptake of 104 pg C cell1 h1 at approximately 80 mE2 s1. This is well in accord with our results with the Type II D. acuminata populations that had low/lowered maximum productivity. Gisselson et al. (2002) estimated growth rates of D. norvegica populations in 1998 and 1999 with flow cytometric measurements of DNA cell cycle and concluded on the basis of their previous P–E measurements that photosynthesis alone was sufficient to support the growth rates measured in 1998 (0.10– 0.17 day1), but not in 1999 (maximum 0.4 day1). The low productivity values in 1998 were attributed to turbulence-induced disturbance and nutrient deficiency (not measured in 1999). Mixotrophy was
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suggested to explain the discrepancy between growth rates based on photosynthetic carbon uptake rate (P–E measurements) and those based on DNA cell cycle analysis in 1999, although only 10% of the cells in that year showed signs of heterotrophy (feeding vacuoles). The P–E incubations in our data set show the variability in the production efficiency of different D. acuminata populations. If this holds for Baltic D. norvegica populations as well, the low photosynthetic production rates obtained by Gisselson et al. (2002) might be explained by the life history status of the particular population used in their measurements. Light levels at the thermocline do not support efficient autotrophic growth of Dinophysis spp. (Carpenter et al., 1995; Gisselson et al., 2002, this study). In order to grow autotrophically, the thermocline populations would need to migrate to higher light levels. Dinophysis acuminata performs phototactic vertical migrations in some environments (e.g. Maestrini et al., 1995; Villarino et al., 1995; Figueroa et al., 1998). In a shallow, well-stratified estuary, the vertical migration of D. acuminata was negatively phototactic, approximately 6 m in distance, and began under conditions of high irradiance (Figueroa et al., 1998). Maestrini et al. (1995) used the phototactic migration as a tool to separate Dinophysis spp. cells from sinking material. The migrational patterns of dinoflagellates may be related to various environmental factors, including light intensity and nutrient availability (Cullen and MacIntyre, 1998). Phototactic migration can be positive or negative, and triggered by a certain level of incident irradiance. Certain dinoflagellate species perform vertical migration in response to nutrient depletion. Cullen and Horrigan (1981) found Gymnodinium splendens descended to 18 m to the top of a nitracline during the night, and ascended during the day to the depth of maximal photosynthetic rate. In a recent study in the northern Baltic Sea, Kononen et al. (2003) found a deep-water chlorophyll maximum formed by vertical migration of the bloom-forming dinoflagellate Heterocapsa triquetra. The authors concluded that H. triquetra population experienced an unbalanced N:P ratio after an upwelling event that provided phosphate to the productive water column. The nitrogen-depleted H. triquetra population migrated from the nitrate depleted euphotic zone down to the top of the nitracline at 30–35 m depth, where only <0.1% of
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surface illumination was available, and remained there for 3–6 days. When the survival of H. triquetra cells in darkness was experimentally studied, they maintained their photosynthetic capacity and approximately half of their initial abundance during an 18 day incubation. In the few field studies made on Dinophysis species in the Baltic Sea, vertical migration has not been observed (Carpenter et al., 1995; Gisselson et al., 2002). Carpenter et al. (1995) intensively sampled D. norvegica for 2–3 days at the same site, and would probably have detected diel migrational patterns, if they occurred, but not necessarily such a prolonged migration as found for H. triquetra by Kononen et al. (2003). Our 2001 laboratory incubations showed the ability of D. acuminata cells to persist for a relatively long time at a light level that approximated 3% of the incident light level (i.e. 10 m depth). The P–E incubations of the deep-water populations and the artificially darkened population verified that the cells were still able to photosynthesize. This could mean that D. acuminata cells have the ability to persist in low light for several weeks, and if dispersed again to upper water layers, continue photosynthesis, although at a lower rate. The recovery of their photosynthetic capacity was, however, not followed in our study. If it is assumed that the incident irradiance approximates 1500 mE m2 s1, and the average attenuation coefficient (kd) is 0.50 (J. Seppa¨ la¨ , Finnish Institute of Marine Research, unpublished, measured at an opensea station on the SW coast of Finland in August), then the light level for the maximum photosynthetic carbon uptake rates of Dinophysis acuminata measured in the present study would occur between 2.6 (500 mE m2 1 2 1 s ). Dinoflagellates s ) and 3.6 m (250 mE m are usually slightly more dense than seawater, and their swimming speeds generally vary between <0.1 and >0.6 mm s1 (Kamykowski and Mc Collum, 1986; Levandowsky and Kaneta, 1987). Dinophysis spp. are presumably not the fastest swimmers among dinoflagellates, but if they were able to swim with the speed of 0.5 mm s1, they would pass the distance from 14–15 to 2–3 m in about 5–6 h. From 20 m depth the journey to the optimal light conditions would take considerably longer. At this speed, the time to traverse the distance between 80 m and surface is 44 h, assuming motility to be more or less continuous and that the cells are able to pass density gradients.
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The cellular carbon content of an average sized Dinophysis acuminata cell (15 900 mm3) calculated after the conversion presented in Menden-Deuer and Lessard (2000) would be 2098 pg. Assuming a growth efficiency of 40% and maximum photosynthetic carbon uptake rate for D. acuminata equal to 925 pg C cell1 h1, it would take approximately 5.8 h to produce the amount of carbon needed for cell division. This means that under optimal conditions the journey from the thermocline to Pmsaturating light conditions and the time needed to produce phototrophically enough carbon reserves for the formation of a daughter cell would take 10–12 h. The Dinophysis spp. community in year 2001 occurred in a water layer where the nutrient concentrations peaked. Below that, the oxygen concentrations declined. This might indicate that this Dinophysis population had migrated down to optimal conditions to renew their nutrient reserves. Motility upwards would have offered lower nutrient concentrations while deeper migration would be prevented by the lowered/exhausted oxygen concentration met already at 80 m (0.7 ml l1). The lack of other organisms in that layer supports the idea of an active migration, and means also that the survival of D. acuminata in darkness was not supported by phagotrophy, since only very few protist as prey candidates were found.
5. Conclusions This study revealed the in situ occurrence of Dinophysis acuminata in conditions unsuitable for phototrophic nutrition, and an ability to persist in low light and temperature for several weeks. Experimental exposure to other protists did not have any clear positive effect on the survival of D. acuminata. On two occasions, a D. acuminata cell was found attached to a small cell with a thin cytoplasm strand, a finding that could indicate feeding, i.e. mixotrophy. The P–E incubations in 2002 showed the D. acuminata populations studied to be photosynthetically active. The maximum photosynthetic productivity of different populations was reached at 250– 500 mE m2 s1 but with differences in the Pm rates that seemed related to the depth from which the cells originated. The photosynthetic carbon production rates of the artificially darkened population and the
two populations that originated below the thermocline were lower than in the populations originating from upper water layers. The photosynthetic activity of D. acuminata cells collected from deep layers and their capability to survive for several weeks on their cellular reserves indicate their recent descent to deep water layers, and possibly a characteristic phenomenon in some Baltic Sea Dinophysis spp. communities. Certain Dinophysis spp. communities in the northern Baltic Sea may thus be able to actively migrate to nutrient-rich water layers to renew their cellular nutrient reserves. However, the causes of the occurrence of Dinophysis acuminata cells in the deep-water layers is not solved by this study, although it was probably due to some recent event. Our findings suggest that the occurrence of the D. acuminata cells in deep water layers may have been due to active, prolonged migration triggered by some environmental factors, such as high nutrient concentrations in the deep layers.
Acknowledgements We thank Dr. Diane Stoecker, Dan Gustafson and Matt Johnson from Horn Point Environmental Laboratory (UMCES) for providing us with strains of planktonic algae and ciliates, and Dr. Wayne Coats from Smithsonian Environmental Research Center (SERC) for his help with the analysis of D. acuminata cell contents. We also thank the staff of Finnish Institute of Marine Research for their assistance onboard R/V Aranda, and Mervi Sjo¨ blom and Elina Salminen (Tva¨ rminne Zoological Station) for nutrient analyses. Maija Huttunen (Finnish Institute of Marine Research) helped us with the Dinophysis spp. identification, and Jukka Seppa¨ la¨ with estimates of the light attenuation. Thanks also to Pekka Punttila (Finnish Environment Institute) for valuable comments and corrections. The Walter and Andre´ e de Nottbeck Foundation and the Finnish Institute of Marine Research supported this work.
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Survival and photosynthetic activity of different Dinophysis acuminata populations in the northern Baltic Sea Outi Seta¨la¨a,b,c,*, Riitta Autiob, Harri Kuosac, Janne Rintalab,c, Pasi Ylo¨stalob a
Finnish Environment Institute, Mechelininkatu 34a, P.O. Box 140, FI-00251 Helsinki, Finland b Finnish Institute of Marine Research, P.O. Box 33, FI-00931 Helsinki, Finland c Tva¨rminne Zoological Station FI-10900 Hanko, Finland Received 25 February 2004; accepted 14 June 2004
Abstract This study deals with a recently found phenomenon in the northern Baltic Sea: the occurrence of the dinoflagellate Dinophysis acuminata in the deep water below the thermocline. This was first observed in July 2001 at the station BY 15 in the Gotland Deep, where a sharp and intensive chlorophyll fluorescence signal was encountered at 77 m depth. The fluorescence peak was due to a dinoflagellate community dominated by Dinophysis acuminata (approximately 18 000 cells l1). The survival of this community was followed in laboratory incubations in low light (20 mE m2 s1) and low temperature (+5 8C). After 5 weeks incubation, 67–84% of the initial cell abundance was lost, while few D. acuminata cells survived up to 24 weeks in the original sample. During the incubation, the fluorescence signal of the cells became fainter and the chloroplasts smaller and aggregated. On two occasions a D. acuminata cell was found attached to a smaller cell by a thin cytoplasm strand, possibly indicating mixotrophic behavior. During the following summer (2002), the photosynthetic efficiency of D. acuminata collected from thermocline layers of few stations and from the nitracline (75–80 m) at one station was studied in photosynthesis irradiance (P–E) incubations. Photosynthetic activity occurred in all populations, with differences in their photosynthetic carbon uptake rates. Photosynthesis of D. acuminata populations was saturated between 250 and 500 mE m2 s1; maximum cell-specific carbon uptake rates (Pm) ranged from 160–925 pg C cell1 h1. The Pm-rates in populations originating below the thermocline and in an artificially darkened population were markedly lower than in populations from upper water layers. The varying maximum photosynthetic rates of these populations may reflect their history, e.g. time spent in different light environments. # 2004 Elsevier B.V. All rights reserved. Keywords: Survival; Carbon uptake; Vertical migration
1. Introduction * Corresponding author. Tel.: +358 9 40300292; fax: +358 9 40300291. E-mail address: [email protected] (O. Seta¨la¨).
Dinophysis acuminata and Dinophysis norvegica are common and frequently abundant dinoflagellate species in the northern Baltic Sea. These species often
1568-9883/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2004.06.017
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dominate the late summer algal blooms in the Gulf of Finland (Kononen and Niemi, 1986; MeyerHarms and Pollehne, 1998; Kononen et al., 1999, 2003; Gisselson et al., 2002). In the brackish central and northern Baltic, they occur usually at concentrations of 102–103 cells l1 (Carpenter et al., 1995; Gisselson et al., 2002; Salomon et al., 2003), and occasionally reach higher concentrations of up to 150 103 cells l1 (Carpenter et al., 1995). When abundant in late summer and autumn, autotrophic Dinophysis spp. are regularly found concentrated at the thermocline in 15–25 m (Niemi, 1975; Kuosa, 1990; Carpenter et al., 1995; Gisselson et al., 2002). The relationship between Dinophysis blooms and the environmental factors has been studied by several authors (e.g. Delmas et al., 1992; Subba Rao et al., 1993; Carpenter et al., 1995; Figueroa et al., 1998; Gisselson et al., 2002; Godhe et al., 2002). The conditions leading to Dinophysis spp. blooms are of special interest, since many Dinophysis species produce toxins that may cause severe economical problems in mussel farming areas (Hallegraeff, 1995). In the Baltic Sea okadaic acid, a toxin produced by Dinophysis species, has been observed in the soft tissues of blue mussel (Pimia¨ et al., 1998) and in flounder liver (Sipia¨ et al., 2000). Many studies have shown the connection between high abundances of Dinophysis spp. and stratification (e.g. Lassus et al., 1988, 1991; Delmas et al., 1992; Reguera et al., 1995). On the other hand, no evidence of blooms promoted by elevated nutrient concentrations has been found. Instead, both thermal (Delmas et al., 1992) and salinity induced stratifications (Giacobbe et al., 1995; Peperzak et al., 1996; Godhe et al., 2002) have been associated with high Dinophysis abundances. Dense blooms of Dinophysis spp. along the French Atlantic coast were observed both in nutrient-poor and nutrient-rich waters, and the main factor promoting Dinophysis spp. growth was concluded to be thermal stratification that lasted for over 2 weeks (Delmas et al., 1992). In Bedford Basin, eastern Canada, D. norvegica bloomed when a strong density gradient suppressed turbulence allowing the cells to concentrate (Subba Rao et al., 1993). Neither study found that Dinophysis blooms would depend on the availability of dissolved nutrients.
Autotrophic dinoflagellates perform diel vertical migration (DVM) in the water column that is typically induced by taxic responses to light and/or gravity. Changes in the biochemistry over the cell cycle (Kamykowski, 1995), cellular nutrient ratios (Kononen et al., 2003) and chemical and thermal gradients (Cullen and Horrigan, 1981; Cullen, 1985; MacIntyre et al., 1997) influence the distribution and migration patterns of dinoflagellates. The DVM patterns express near-surface aggregations during daylight with descent or dispersal during the night. In the Baltic Sea, the Dinophysis spp. occurring at relatively low light environments at the thermocline have not been observed to perform vertical migrations. This has raised questions about their ability to provide carbon for their growth by photosynthesis in environments where light supply is limited (Carpenter et al., 1995; Gisselson et al., 2002). In Ria de Vigo, NW Spain, Dinophysis acuminata has, however, been found to migrate vertically between surface and 10 m depth (Villarino et al., 1995). In the same area, Figueroa et al. (1998) found D. acuminata cells aggregated close to the surface at noon and descended during the night. In this shallow stratified estuary, the cells, however, were seemingly unable to swim through a well-defined pycnocline. Attempts to culture Dinophysis spp. in various culture media, or with accompanying phytoplankton species, have shown an ability of the cells to survive on their own cellular reserves for a few weeks, and, at best, to grow and persist for 16–20 weeks (e.g. Nishitani et al., 2003; Sampayo, 1993). Generally the cells become thinner and less pigmented with time, with reduced and deformed plasmatic contents (Sampayo, 1993; Maestrini et al., 1995, this study). So far, all attempts to establish a true culture of Dinophysis spp. in media that support the growth of other phytoplankton species have failed (Keller and Guillard, 1985; Sampayo, 1993; Maestrini et al., 1995; Subba Rao, 1995; Grane´ li et al., 1997; Nishitani et al., 2003). A routine CTD-cast in the Baltic Proper revealed a sharp chlorophyll fluorescence signal at 77 m. The water was found to contain a dense community (approximately 18 103 cells l1) of Dinophysis spp. at a depth where no light to support photosynthesis is available. Preliminary studies (laboratory incubations) were conducted to follow the survival capacity
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of the D. acuminata cells in this deep-water community. During the following summer (August 2002) the studies were continued by evaluating the photosynthetic activity of cells collected below the thermo- and halocline from open sea, deep-water stations and from the thermocline of one coastal sampling site.
2. Material and methods 2.1. Sampling Most of the material for this study was collected in the Gotland Sea onboard R/V Aranda (Finnish Institute of Marine Research) during two research cruises in August 2001 and 2002. The data set for year 2001 includes the first observation of a deep-water community of Dinophysis spp. from a Gotland Sea station (BY 15, the Gotland Deep), and the laboratory incubations. The data set from August 2002 includes the P–E measurements of different Dinophysis acuminata populations collected from an open-ocean station in the northern Baltic Sea (LL 23; total depth 459 m) and from a coastal station (Tva¨ rminne Storfja¨ rd, total depth 33 m). Temperature, salinity and the in situ Chl-a fluorescence were measured with a SBE 911 Plus CTD-probe (SeaBird Electronics) equipped with a SeaTech (440/680) fluorescence sensor. Dissolved inorganic nutrients, oxygen, H2S and Chl-a concentrations were analyzed according to standard methods (Grasshoff et al., 1983). Temperature, salinity and nutrient profiles from Tva¨ rminne Storfja¨ rd are presented for the days of measurement closest to the actual sampling dates of D. acuminata populations. Samples for phytoplankton counts were preserved with acid Lugol’s solution (Ha¨ llfors et al., 1979), and settled in 50 ml tubes for 24 h following Utermo¨ hl (1958), and counted with inverted microscope (Leica DMIL, 100–400 magnifications). Samples were also fixed with 25% glutaraldehyde (final conc. 1–2%) and studied with Leica DMIL inverted microscope with epifluorescence setup under green (546/14 nm, 580 nm beam splitter, 590 nm barrier filter) and blue excitation light (450–490 nm, 510 nm beam splitter, 515 nm barrier filter), in order to study the condition and distribution of chloroplasts of the cells.
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2.2. The survival of Dinophysis acuminata in laboratory incubation As a pilot study, the viability of the cells was studied in laboratory incubations. Two 8 l polycarbonate bottles were filled with water collected from 77 m at the Gotland Deep station (year 2001), and kept in an incubation room (+3 8C, darkened) onboard R/V Aranda for 7 days (until the end of the cruise). Further incubations were made in an onshore laboratory in four 4 l polycarbonate bottles, each containing 3.5 l of water and kept at +5 8C exposed to a 12:12 h light:dark cycle under 20 mE m2 s1 with minimal mechanical disturbance. Since the concentrations of the inorganic nutrients at the start of the incubation were high (NH4–N: 1.2 mmol l1, NO3 + NO2–N: 9.4 mmol l1, PO4–P: 2.8 mmol l1), nutrient additions were not made. Because D. acuminata occurred in darkness, mixotrophy was speculated to play a role in their nutrition. Live, cultured microalgae and the ciliate Mesodinium pulex were added to the incubated samples (=units) at low concentrations (algae: 250– 400 cells ml1, M. pulex: 5 cells ml1) to see whether D. acuminata would benefit from the presence of other protists. The accompanying species were: Heterocapsa triquetra and Heterocapsa rotundatum (unit B = dinoflagellate unit), Prorocentrum minimum, Storeatula major and Mesodinium pulex (unit C = mixed unit), Storeatula major and Pyrenomonas sp. (unit D = cryptophyte unit). Unit A was the untreated control. The added algae had been grown under 12:12 h light:dark cycle (40–60 mE m2 s1) at 18 8C in f/2 (– Si) medium (Guillard, 1975). The ciliate Mesodinium pulex had been cultured in a seawater medium containing 0.1 ml l1 of f/2 iron-EDTA trace metal solution (Guillard, 1975) with Storeatula major as prey. The survival of D. acuminata cells in each treatment was followed weekly by examining live samples under stereomicroscope (6.3–50 magnification, Leica MZ 7.5) to assess their condition, and to obtain a rough estimate of their abundance. This was done by carefully pouring water into a petri dish and letting it settle for approximately 15 min, after which swimming D. acuminata cells were easily detected. Samples for more detailed microscopy examinations were fixed with glutaraldehyde or with acid Lugol’s solution (Ha¨ llfors et al., 1979). Regular quantitative
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sampling was not considered, since it would have consumed too much of the experimental water; therefore, the outcome of these incubations was solely observational. 2.3. P–E measurements Photosynthetic carbon uptake rates (P–E measurements) of different D. acuminata populations were measured in August 2002. Since the cells were dispersed in the water column in abundances of 1–2 103 cells l1, the water was concentrated following Maestrini et al. (1995). The water sample was successively poured through two different mesh sizes (100 and 20 mm) and the filtrate gently siphoned. If the filtrate contained a marked number of cells other than Dinophysis, it was further filtered through a 30–50 mm net, using an inverse method in which water is carefully sucked through the filter with a syringe. With this procedure, the D. acuminata cells were left in the water and smaller cells excluded. If the ciliate Mesodinium rubrum was abundant in the sample, which often was the case, the water was gravity filtered through a 63 mm net, a method that eliminates the fragile M. rubrum cells which explode during filtration, but allows most of the D. acuminata cells to pass through undamaged. The filtrate was settled for 2–24 h, after which the upper part of the water was gently poured into a 1–2 l polycarbonate bottle. This allowed only actively swimming cells to be incubated. A 5–10 ml sample from the filtrate was fixed with Lugol’s solution, pipetted into Utermo¨ hl chambers, and the number of cells in the sample counted. This filtration protocol yielded concentrations of 30–160 D. acuminata cells ml1 for the P–E measurements. The cells from the open-sea station, LL 23, were sampled at the thermocline at 14 m and from deep water at 80 m. Two sets of samples were collected at the coastal station, Tva¨ rminne Storfja¨ rd, from the thermocline at 15–17 m and from 20–22 m (2 and 7 August). All P–E incubations were done on the sampling day, except the other half of the cells from Tva¨ rminne Storfja¨ rd sampled on 2 August, that were incubated for 6 days in darkness (in situ temperature) before measurement. Photosynthetic carbon uptake was measured as 14 C-CO3 incorporation in a P–E incubator with 16 different light levels, and 2 dark incubations serving as
controls. The light levels were provided with a 300 W quartz halogen lamp covered with heat shields and with an output of 2475 mE m2 s1 and measured with a scalar irradiance meter (Biospherical Instruments, QSL-2101). Duplicate sets of the D. acuminata concentrates (volume 3 ml) were incubated for 2 h in 7 ml glass scintillation vials. One microcurie of NaH14CO3 (DHI Water & Environment, Denmark) was added to the samples prior to the incubation, which were terminated by adding 100 ml of 38% formaldehyde to the samples. Net primary productivity was measured by removing the remaining inorganic carbon by adding 100 ml 1N HCl to the vials and letting them stand open for 48 h. After the acidification, 4 ml of scintillation cocktail (Instagel+, Packard, USA) was added to the vials and the radioactivity measured with a scintillation counter (LKB Wallac RackBeta, Finland). The average DPM count value of duplicate dark bottles was subtracted from each sample. Cell-specific carbon uptake was calculated as P ðDPMcell DPMdark Þð CO2 ÞVk1 P¼ DPMadded T where P is the cell-specific carbon uptake, DPMcell the DPM count from a sample divided by the number of D. acuminata cells in that sample, P DPMdark the DPM/cell from the dark controls, CO2 the total dissolved inorganic carbon in the water, V the sample volume (3 ml), k1 the correction factor for isotope discrimination (1.05, Æertberg-Nielsen and Bresta, 1984), DPMadded the DPM added to each vial, and T the incubation timePin hours. In brackish waters, such as the Baltic Sea, CO2 can be calculated according to the formulas of Buch (1945). The P–E parameters for the comparison of different curves were calculated using a modified version of the exponential model according to Platt et al. (1980): B B a E b E B B P ¼ Ps 1 exp exp PB PB s s where PB [pg C cell1 h1] is the cell-specific rate of carbon incorporation at irradiance E, aB [(pg C cell1 h1) (mE m2 s1)1] the initial slope of the P–E curve, E the irradiance [mE m2 s1] and bB [pg C (cell1 h1) (mE m2 s1)1] the photoinhibition parameter, PB s the equivalent to the maximum
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rate of cell-specific rate of carbon incorporation, PB m [pg C cell1 h1], when bB = 0. Otherwise when bB 6¼ 0, realized cell-specific maximum rate of carbon incorporation, PB m is calculated according to Platt et al. (1980) as follows: PB m
¼
PB s
aB a B þ bB
bB a B þ bB
b=a
The light saturation parameter, Ek [mE m2 s1] is B calculated as Ek = PB m /a . 3. Results 3.1. Hydrography, inorganic nutrients, Chl-a and plankton community In 2001, a well-defined thermocline was situated at 13–17 m depth at station BY 15. The deeper water column was separated by a halocline at 60–80 m (Fig. 1). The oxygen concentration declined rapidly below 70 m, and traces of H2S were detected at 125 m and below. The concentrations of inorganic nutrients in the water column above thermocline were low (PO4–P and NO3 + NO2–N: <0.05 mmol l1, NH4–N: <0.20 mmol l1), but increased markedly below 50 m. The highest concentration of inorganic nitrogen was measured at 80 m depth (NO3 + NO2–N: 9.4 mmol l1). Chl-a concentrations peaked at 10 m depth (2.23 mg Chl-a l1) and again at 77 m (0.92 mg Chl-a l1). The measured hydrographical parameters and inorganic nutrient concentrations at station LL 23 in year 2002 were similar to those described for the Gotland Deep (BY 15) in the previous year, except for somewhat higher temperatures. The hydrography of the coastal station, Tva¨ rminne Storfja¨ rd, reflected warm and calm weather conditions. The salinity was approximately 5 psu through the whole water column, and temperature was >20 8C down to the thermocline at 15–17 m. The inorganic nutrient concentrations increased with depth. In year 2001, the phytoplankton community in the euphotic layer consisted mainly of small, <10 mm flagellates. Filamentous blue-green algae were practically absent. Dinoflagellates formed 2.8–9.8% of the total autotrophic phytoplankton biomass in 0–12 m.
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Dinophysis spp. were absent, or in low abundance at all depths except at 77 m, where 18 103 cells l1 (37.8 mg C l1) were found. Approximately 95% of the cells were identified as Dinophysis acuminata, and the rest were D. norvegica (M. Huttunen, Finnish Institute of Marine Research, (pers. comm.). The phototrophic ciliate Mesodinium rubrum was abundant in the upper water column (2.4–6.1) 103 cells l1). The phytopankton community in the euphotic zone in the year 2002 sample consisted mostly of large colonial or filamentous blue-green algae, (<3 mm) and auto- and heterotrophic flagellates. The proportion of blue-green algae above the thermocline increased with depth. As in 2001, the proportion of dinoflagellates, including Dinophysis spp., was insignificant in the 0–12 m water layer. Dinophysis acuminata was found in low concentrations (<2 103 cells l1) between 70 and 80 m, as was M. rubrum (<100 cells l1). The phytoplankton community in year 2002 in Tva¨ rminne Storfja¨ rd consisted of filamentous blue-green algae (mainly Aphanizomenon flos-aquae and small Pseudoanabena species) and small autotrophic flagellates (Chrysochromulina spp., Pseudopedinella spp., Cryptomonadales and <3 mm unidentified cells). The phytoplankton community at the thermocline region was similar, except for declining abundance of Aphanizomenon flos-aquae and the presence of Dinophysis spp. 3.2. Laboratory incubations The numbers of D. acuminata cells decreased in all experimental treatments during the first week. On the fifth laboratory incubation week (7 weeks after initial sampling from 77 m), 67–84% of the original cell abundance had been lost, and 3–6 cells ml1 in three of the four units were found. In experimental unit B the vigorous growth of Heterocapsa spp. during the first 2 weeks of incubation, and the successively decaying phytoplankton material either overgrew D. acuminata or masked it, since living D. acuminata cells were not found in this unit. Motile D. acuminata cells were abundant on week 7 in units A (control) and D (cryptophyte), whereas only few cells were routinely found in the mixed population unit C. Between weeks 7 and 16, swimming cells were found in units A and D, but only occasionally in unit C. On
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Fig. 1. (A–I) Profiles of temperature, T (8C), salinity, S (psu), oxygen, O2 (ml l1), nutrient concentration (mmol l1), Chl-a (mg l1), CTD fluorescence (arbitrary units) and D. acuminata biomass (mg C l1) at the sampling stations in years 2001 and 2002.
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week 16 of the experiment incubation of the units C and D was terminated since no living D. acuminata cells were observed. In February 2002, 6 months after the initial sampling, few slowly swimming D. acuminata cells were still found in the control unit. Dividing cells were not observed in any treatment during the incubations.
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distributed and tightly packed, as described above, and more or less rounded vacuole-like bodies were observed. These bodies were of different sizes and transparent under transmitted light. Occasionally, a whole cell was green under blue excitation light (Fig. 3D). On two occasions a Dinophysis acuminata cell was found attached to a small (approximately 7– 8 mm) cell by a thin cytoplasm strand (Fig. 2E and F).
3.3. Observations on Dinophysis acuminata cell structure
3.4. Photosynthesis
In the beginning of the incubation period, the cells were mostly in good condition, with branched, brightly fluorescent yellowish-orange chloroplasts. Thereafter the chloroplasts of the incubated cells generally became smaller and densely packed (Fig. 3A and B), the fluorescence signal became fainter, and finally (after 6 months incubation) only one or two ‘‘packages’’ of faintly coloured chloroplasts in otherwise transparent cells were seen (Fig. 3C and D). Various types of ‘‘deformations’’ in the cell contents were seen with epifluorescence microscopy from week 5 onward. The chloroplasts were unevenly
All D. acuminata populations were photosynthetically active. The P–E parameters and the corresponding P–E curves from the incubations for each population are presented in Table 1 and Fig. 2. The populations could be divided into two types based of their specific P–E characteristics and are referred to as the Type I and Type II populations. Type I includes photosynthetically ‘‘very active’’ populations (Tva¨ rminne Storfja¨ rd: 15–17 m and LL 23: 14 m), with light-saturated maximum photosynthetic carbon uptake (Pm) of 782 and 925 pg C cell1 h1. The a values describing the light limited initial slope were
Fig. 2. (A–E) P–E curves for different D. acuminata populations in year 2002. (A–C) Tva¨ rminne Storfja¨ rd (Tv): A = thermocline, B = the population in A after 6 days dark incubation, C = below thermocline. (D, E) Landsort deep (LL 23): D = thermocline region, E = deep-water layer. For the curve fitting see Section 2.
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Table 1 P–E parameters of the Dinophysis acuminata populations measured in August 2002 Station
a b Pm Ek
Tv 2.8. (depth = 15–17 m)
Tv 2.8. (depth = 15–17 m, darkened)
Tv 7.8. (depth = 20–22 m)
LL 23 14.8. (depth = 14 m)
LL 23 14.8. (depth = 80 m)
6.29 0.22 783 124
1.90 0.56 361 190
2.11 0.19 210 100
9.74 1.12 925 95
2.19 0.06 160 72
Tv = Tva¨ rminne Storfja¨ rd, LL 23 = Landsort Deep. Description of the parameters: a = light-limited initial slope [(pg C cell1 h1) (mE m2 s1)1], b = parameter describing the reduction in photosynthetic rate with high irradiance [pg C (cell1 h1) (mE m2 s1)1], Pm = light-saturated maximum rate of photosynthesis (pg C cell1 h1), Ek = light saturation parameter (mE m2 s1).
high (6.29 and 9.74) in these two populations collected closest to the surface, indicating a rapid increase in the production rate with increasing irradiance. The other three populations (Type II populations) were characterized by low/lowered photosynthetic activity, expressed as low Pm (160–361 pg C cell1 h1) and a (1.90–2.19) values. The two measurements made with the populations from below the thermocline (Tva¨ rminne Strorfja¨ rd: 20–22 m and LL 23: 80 m) had the lowest Pm values (Fig. 2C and E). The population collected from Tva¨ rminne Storfja¨ rd from 15–17 m depth and kept in darkness for 6 days prior to the P–E incubations had somewhat higher measured Pm levels than the deep-water samples (Table 1, Fig. 2B). In all populations, photosynthesis was saturated at irradiances between ca. 250 and 500 mE m2 s1. The b value describing photoinhibition was highest in the 14 m population from open-sea station LL 23. The dark uptake rates of 14C were <10% of the maximum uptake rates in all incubations.
4. Discussion The Baltic Sea is characterized by a high concentration of coloured dissolved organic matter that affects the attenuation of solar irradiance. As a consequence of the overall high light attenuation in the Baltic Sea, the euphotic zone (>1% of surface light, PAR) does not exceed 20–30 m (e.g. Aarup, 2002). Because of these properties, the solar irradiance in the northern Baltic does not penetrate below the halocline, which also means that the Dinophysis spp. cells collected from 77 and 80 m evidently occurred in
darkness. The occurrence of these cells in such an extreme and isolated environment suggests that these species may migrate through strong gradients, and persist at conditions unsuitable for any active and primarily phototrophic organism. The laboratory incubations of this study showed that a part of the deep-water Dinophysis acuminata population was able to survive in rather extreme conditions for up to 6 months, although in the end only few active cells were found. Their survival indicates the ability of D. acuminata to persist for a relatively long time period in unfavorable conditions. This is of special interest in light of the difficulties met in earlier culturing attempts. Recently, Nishitani et al. (2003) tried to cultivate Dinophysis caudata together with accompanying phytoplankton (Thalassiosira sp., Chroomonas sp. and an eukaryotic picoplankton species) as possible prey. At best, they were able to grow 28 cells from one D. caudata cell, and keep them alive up to 2 months, which may indicate that D. caudata benefited from the addition of other protists. This, however, does not conform to our observations of D. acuminata survival in the different experimental units, where no clear benefit of the accompanying species was found. One reason for successful growth in the study by Nishitani et al. (2003) could have been that the algae were added crushed and frozen before addition, and thus did not compete with D. caudata cells. Our incubations revealed that the size and shape, and the fluorescence intensity of the chloroplasts diminished with time; the last observed living cells were almost transparent (Fig. 3C). Lucas and Vesk (1990) suggested that in recently divided cells the
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Fig. 3. (A–F) Various types of Dinophysis acuminata cells. (A, B) Cells with beady chloroplasts, (C, D) cells near termination of incubation with only few packages of chloroplasts, (E, F) a D. acuminata cell attached to a smaller cell. (A, C, E) Light microscopy, (B, D, F) epifluorescence microscopy.
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chloroplasts appear scattered through the cytoplasm, whereas in older cells they aggregate into clumps. The chloroplasts of the cells collected from the deep-water layers during both years appeared, however, to be mostly typical of those for healthy, and presumably recently divided cells (bright and branched). If the recorded changes in the size and shape of the chloroplasts were typical and related to their aging process, then the occurrence of the Dinophysis spp. cells in the deep-water samples must have been the result of some relatively recent process. Mixotrophy by phagotrophy or osmotrophy has been suggested to be an important factor supporting the nutrition of Dinophysis spp. (e.g. Jacobson and Andersen, 1994; Carpenter et al., 1995; Grane´ li et al., 1997). Jacobson and Andersen (1994) found food vacuoles inside D. acuminata and D. norvegica, which was first-hand evidence of their phagotrophic capability. Based on the food vacuole contents, these authors suggested that these autotrophic Dinophysis spp. were preying on ciliates in the same manner as the heterotrophic Dinophysis rotundata observed to feed on ciliates of their own size (Hansen, 1991). Nishitani et al. (2002) observed picoplanktonic algae attached to the surface of Dinophysis spp. and food vacuole-like structures filled with red fluorescence probably from algal prey. Although no clear relationships between the abundance of picoplankton and Dinophysis spp. were detected, Dinophysis spp. were suggested to feed on them. Signs of mixotrophy was also found by Ishimaru et al. (1988), who observed how attached cryptophytes were absorbed inside Dinophysis fortii. Our images of D. acuminata cells in contact with a small cell can be interpreted in several ways. Both Subba Rao (1995) and Berland et al. (1995) have reported the development of unknown, round bodies inside D. acuminata cells, and their release. Some of these bodies were packed with small granula, suspected to be zooids or gametes. Our images of the two connected cells resemble those illustrated by Subba Rao (1995). However, the vacuole-like bodies that we observed inside D. acuminata were of various size and shape, and we did not observe gametes or zooids. The occurrence of deformed, beady-looking chloroplasts in Dinophysis spp. has been noted by several authors. Noren et al. (1999) reported granular chloroplasts in D. norvegica cells infected by the parasite flagellate Parvilucifera infectans. Infection of
D. norvegica populations by another parasite, Amoebophrya sp. trophonts was reported by Gisselson et al. (2002) and Salomon et al. (2003). Our Dinophysis acuminata populations also showed signs of Amoebophrya infections, but the prevalence appeared low (approximately 5%; Wayne Coats, Smithsonian Environmental Research Center, pers. comm.). It remains unclear whether the connected Dinophysis acuminata cells were releasing gametes/zooids, under a parasite attack, or actually feeding on the smaller cells. Since the photosynthetic activity of the Dinophysis spp. community in year 2001 sample was not measured, the ‘‘condition’’ or viability of the cells was estimated by microscopical observations only. However, the P–E measurements made in the following year showed that the deep-water community consisted of active cells. Populations originating from different depths varied in their maximum photosynthetic carbon uptake rates (Pm values). The lowest Pm values were obtained from the populations collected below the thermocline, and were lower than the values of the artificially darkened population. This may reflect the history of these populations, i.e. the longer the time period spent in darkness, the lower the Pm value. In the two populations from the thermocline layer, the maximum photosynthetic carbon uptake rates were markedly higher (Pm: 783 and 925 pg C cell1 h1). Photosynthetic carbon uptake rates of Dinophysis norvegica were measured by Gisselson et al. (2002) using light levels of 8– 330 mE2 s1. Their measurements were done with one population collected from 18 m depth, and gave saturated carbon uptake of 104 pg C cell1 h1 at approximately 80 mE2 s1. This is well in accord with our results with the Type II D. acuminata populations that had low/lowered maximum productivity. Gisselson et al. (2002) estimated growth rates of D. norvegica populations in 1998 and 1999 with flow cytometric measurements of DNA cell cycle and concluded on the basis of their previous P–E measurements that photosynthesis alone was sufficient to support the growth rates measured in 1998 (0.10– 0.17 day1), but not in 1999 (maximum 0.4 day1). The low productivity values in 1998 were attributed to turbulence-induced disturbance and nutrient deficiency (not measured in 1999). Mixotrophy was
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suggested to explain the discrepancy between growth rates based on photosynthetic carbon uptake rate (P–E measurements) and those based on DNA cell cycle analysis in 1999, although only 10% of the cells in that year showed signs of heterotrophy (feeding vacuoles). The P–E incubations in our data set show the variability in the production efficiency of different D. acuminata populations. If this holds for Baltic D. norvegica populations as well, the low photosynthetic production rates obtained by Gisselson et al. (2002) might be explained by the life history status of the particular population used in their measurements. Light levels at the thermocline do not support efficient autotrophic growth of Dinophysis spp. (Carpenter et al., 1995; Gisselson et al., 2002, this study). In order to grow autotrophically, the thermocline populations would need to migrate to higher light levels. Dinophysis acuminata performs phototactic vertical migrations in some environments (e.g. Maestrini et al., 1995; Villarino et al., 1995; Figueroa et al., 1998). In a shallow, well-stratified estuary, the vertical migration of D. acuminata was negatively phototactic, approximately 6 m in distance, and began under conditions of high irradiance (Figueroa et al., 1998). Maestrini et al. (1995) used the phototactic migration as a tool to separate Dinophysis spp. cells from sinking material. The migrational patterns of dinoflagellates may be related to various environmental factors, including light intensity and nutrient availability (Cullen and MacIntyre, 1998). Phototactic migration can be positive or negative, and triggered by a certain level of incident irradiance. Certain dinoflagellate species perform vertical migration in response to nutrient depletion. Cullen and Horrigan (1981) found Gymnodinium splendens descended to 18 m to the top of a nitracline during the night, and ascended during the day to the depth of maximal photosynthetic rate. In a recent study in the northern Baltic Sea, Kononen et al. (2003) found a deep-water chlorophyll maximum formed by vertical migration of the bloom-forming dinoflagellate Heterocapsa triquetra. The authors concluded that H. triquetra population experienced an unbalanced N:P ratio after an upwelling event that provided phosphate to the productive water column. The nitrogen-depleted H. triquetra population migrated from the nitrate depleted euphotic zone down to the top of the nitracline at 30–35 m depth, where only <0.1% of
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surface illumination was available, and remained there for 3–6 days. When the survival of H. triquetra cells in darkness was experimentally studied, they maintained their photosynthetic capacity and approximately half of their initial abundance during an 18 day incubation. In the few field studies made on Dinophysis species in the Baltic Sea, vertical migration has not been observed (Carpenter et al., 1995; Gisselson et al., 2002). Carpenter et al. (1995) intensively sampled D. norvegica for 2–3 days at the same site, and would probably have detected diel migrational patterns, if they occurred, but not necessarily such a prolonged migration as found for H. triquetra by Kononen et al. (2003). Our 2001 laboratory incubations showed the ability of D. acuminata cells to persist for a relatively long time at a light level that approximated 3% of the incident light level (i.e. 10 m depth). The P–E incubations of the deep-water populations and the artificially darkened population verified that the cells were still able to photosynthesize. This could mean that D. acuminata cells have the ability to persist in low light for several weeks, and if dispersed again to upper water layers, continue photosynthesis, although at a lower rate. The recovery of their photosynthetic capacity was, however, not followed in our study. If it is assumed that the incident irradiance approximates 1500 mE m2 s1, and the average attenuation coefficient (kd) is 0.50 (J. Seppa¨ la¨ , Finnish Institute of Marine Research, unpublished, measured at an opensea station on the SW coast of Finland in August), then the light level for the maximum photosynthetic carbon uptake rates of Dinophysis acuminata measured in the present study would occur between 2.6 (500 mE m2 1 2 1 s ). Dinoflagellates s ) and 3.6 m (250 mE m are usually slightly more dense than seawater, and their swimming speeds generally vary between <0.1 and >0.6 mm s1 (Kamykowski and Mc Collum, 1986; Levandowsky and Kaneta, 1987). Dinophysis spp. are presumably not the fastest swimmers among dinoflagellates, but if they were able to swim with the speed of 0.5 mm s1, they would pass the distance from 14–15 to 2–3 m in about 5–6 h. From 20 m depth the journey to the optimal light conditions would take considerably longer. At this speed, the time to traverse the distance between 80 m and surface is 44 h, assuming motility to be more or less continuous and that the cells are able to pass density gradients.
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The cellular carbon content of an average sized Dinophysis acuminata cell (15 900 mm3) calculated after the conversion presented in Menden-Deuer and Lessard (2000) would be 2098 pg. Assuming a growth efficiency of 40% and maximum photosynthetic carbon uptake rate for D. acuminata equal to 925 pg C cell1 h1, it would take approximately 5.8 h to produce the amount of carbon needed for cell division. This means that under optimal conditions the journey from the thermocline to Pmsaturating light conditions and the time needed to produce phototrophically enough carbon reserves for the formation of a daughter cell would take 10–12 h. The Dinophysis spp. community in year 2001 occurred in a water layer where the nutrient concentrations peaked. Below that, the oxygen concentrations declined. This might indicate that this Dinophysis population had migrated down to optimal conditions to renew their nutrient reserves. Motility upwards would have offered lower nutrient concentrations while deeper migration would be prevented by the lowered/exhausted oxygen concentration met already at 80 m (0.7 ml l1). The lack of other organisms in that layer supports the idea of an active migration, and means also that the survival of D. acuminata in darkness was not supported by phagotrophy, since only very few protist as prey candidates were found.
5. Conclusions This study revealed the in situ occurrence of Dinophysis acuminata in conditions unsuitable for phototrophic nutrition, and an ability to persist in low light and temperature for several weeks. Experimental exposure to other protists did not have any clear positive effect on the survival of D. acuminata. On two occasions, a D. acuminata cell was found attached to a small cell with a thin cytoplasm strand, a finding that could indicate feeding, i.e. mixotrophy. The P–E incubations in 2002 showed the D. acuminata populations studied to be photosynthetically active. The maximum photosynthetic productivity of different populations was reached at 250– 500 mE m2 s1 but with differences in the Pm rates that seemed related to the depth from which the cells originated. The photosynthetic carbon production rates of the artificially darkened population and the
two populations that originated below the thermocline were lower than in the populations originating from upper water layers. The photosynthetic activity of D. acuminata cells collected from deep layers and their capability to survive for several weeks on their cellular reserves indicate their recent descent to deep water layers, and possibly a characteristic phenomenon in some Baltic Sea Dinophysis spp. communities. Certain Dinophysis spp. communities in the northern Baltic Sea may thus be able to actively migrate to nutrient-rich water layers to renew their cellular nutrient reserves. However, the causes of the occurrence of Dinophysis acuminata cells in the deep-water layers is not solved by this study, although it was probably due to some recent event. Our findings suggest that the occurrence of the D. acuminata cells in deep water layers may have been due to active, prolonged migration triggered by some environmental factors, such as high nutrient concentrations in the deep layers.
Acknowledgements We thank Dr. Diane Stoecker, Dan Gustafson and Matt Johnson from Horn Point Environmental Laboratory (UMCES) for providing us with strains of planktonic algae and ciliates, and Dr. Wayne Coats from Smithsonian Environmental Research Center (SERC) for his help with the analysis of D. acuminata cell contents. We also thank the staff of Finnish Institute of Marine Research for their assistance onboard R/V Aranda, and Mervi Sjo¨ blom and Elina Salminen (Tva¨ rminne Zoological Station) for nutrient analyses. Maija Huttunen (Finnish Institute of Marine Research) helped us with the Dinophysis spp. identification, and Jukka Seppa¨ la¨ with estimates of the light attenuation. Thanks also to Pekka Punttila (Finnish Environment Institute) for valuable comments and corrections. The Walter and Andre´ e de Nottbeck Foundation and the Finnish Institute of Marine Research supported this work.
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