Journal of Ecology and Protection of the Coastline

ISBN 1643-0115

ISBN 1643-0115

© Copyright by Institute of Biology and Environmental Protection of the Pomeranian University in Słupsk

Original research paper

Accepted:

15.05.2014 8.05.2015

PROTOZOAN COMMUNITIES IN THE VISTULA RIVER ESTUARY (BALTIC SEA)

Krzysztof Rychert

, Janina Kownacka

, Magdalena Wielgat-Rychert

, Anna Pluto-Prądzyńska

1

Department of Ecology, Pomeranian University in Słupsk, ul. Arciszewskiego 22b, 76-200 Słupsk, Poland

2

Department of Fisheries Oceanography and Marine Ecology, National Marine Fisheries Research Institute,

ul. Kołłątaja 1, 81-332 Gdynia, Poland

*e-mail: krychert@wp.pl

Abstract

Protozoan communities (heterotrophic nanoflagellates, dinoflagellates, and ciliates) were studied along the Vistula River estuary (southern Baltic Sea) in June 2005. Protozoan biomass ranged from 64.1 to 162 µgC l

close to the river mouth and decreased to 20.7 µgC l

at the most offshore sta- tion. The negative correlation between distance from the mouth and protozoan biomass was highly statistically significant. Within the estuary, the majority of the biomass was contributed by het- erotrophic dinoflagellates and Ebria tripartita (71% on average), whereas heterotrophic nano- flagellates and ciliates contributed 22% and 7% of the protozoan biomass, respectively. At the off- shore station, the contribution of heterotrophic dinoflagellates decreased to typical value of 25%.

The study confirmed elevated significance of heterotrophic dinoflagellates and E. tripartita in the Gulf of Gdańsk. Correlation analysis revealed that all groups of protozoa were significantly and positively related to phytoplankton biomass or primary production, but only heterotrophic ciliates were related to bacterial secondary production.

Key words: heterotrophic protists, nanoflagellates, dinoflagellates, ciliates

INTRODUCTION

In aquatic environments, bacteria and protozoa (= heterotrophic protists) constitute the microbial food web (Azam et al. 1983, Montagnes et al. 2001, Landry and Kirchman 2002, Sherr and Sherr 2002, Dutz and Peters 2008), in which protozoa act as a trophic link between (i) bacteria and small algae (food), and (ii) metazoans such as rotifers and crustaceans (grazers). Protozoan communities comprise heterotrophic

Journal of Ecology and Protection of the Coastline

Vol. 18 pp. 39-53 2014

nanoflagellates, which are mainly bacterivorous and cyanobacterivorous (Fenchel 1982, Sherr and Sherr 2002), and ciliates, which feed on bacteria, algae, and other protozoa (Strom et al. 1993, Sherr and Sherr 2002). In marine environments there is an additional component of the microbial food web, namely heterotrophic dinoflagel- lates (Hansen 1991, Bralewska and Witek 1995, Sherr and Sherr 2002). Dinoflagel- lates ingest algae, protozoa, detrital particles, and even copepod eggs and nauplii (Hansen 1991, Jeong 1999, Sherr and Sherr 2002). Because of the wide range of food they ingest, the protozoan community comprises a complicated web of preda- tor-prey interactions that dissipates most of the energy flowing through microbial food webs (Ducklow et al. 1986). Some protozoa are mixotrophs that rely on both photosynthesis and heterotrophy (Christaki et al. 2009, Esteban et al. 2010), which complicates the analysis of microbial food web functioning.

Protozoan biomass increases along eutrophication gradients (Beaver and Crisman 1982, Arndt 1991). In the marine environment, elevated protozoan biomass is observed in coastal zones and estuaries where water is enriched with nutrients and organic matter of terrestrial origin (Arndt 1991, Setälä and Kivi 2003, Urrutxurtu et al. 2003). The exter- nal input of nutrients fuels primary production (and only indirectly bacterial secondary production via phytoplankton exudates), whereas the load of organic matter constitutes direct food resource for bacteria. As a result, two separate effects are exerted on proto- zoan communities: (i) more food for herbivorous protozoa because of elevated primary production, and (ii) more food for bacterivorous protozoa because of elevated bacterial secondary production (Sherr and Sherr 2002, Urrutxurtu et al. 2003, Rollwagen-Bollens et al. 2011). Each of these effects shape the composition of the protozoan community differently by increasing the biomass of herbivores or bacterivores.

The aim of this study was to assess changes in protozoan community composition along the Vistula River estuary (Baltic Sea). The Vistula River is the second largest river entering the Baltic Sea, with a mean annual discharge of 1 081 m

s

(HELCOM 2004). The study was performed in June 2005. Studies of phytoplankton and bacterial communities conducted simultaneously (Wielgat-Rychert et al. 2013) permitted verification of two alternative hypotheses, namely, whether during the study period, phytoplankton or bacterial production exerted a stronger impact on the composition of the protozoan community.

Three main protozoan groups were studied: heterotrophic nanoflagellates, dinofla-

gellates, and ciliates. The size of the organisms studied spans over a few orders of

magnitude, e.g., the volume of a typical pelagic ciliate is a few hundred times larger

than that of a typical nanoflagellate. Thus, biomass, and not numerical abundance,

was used to assess the relevance of particular protozoan groups within the protozoan

community. Bralewska and Witek (1995), on the basis of studies carried out between

1987 and 1988, indicated high significance of heterotrophic dinoflagellates in the

Gulf of Gdańsk. Their biomass was twice as high as ciliate biomass which is not

a typical ratio for marine systems. Therefore, the secondary aim of this study was to

check whether heterotrophic dinoflagellates were also so important component of

the protozoan communities during our study.

MATERIAL AND METHODS

The study was conducted between June 16

and June 24

, 2005 during a cruise of the r/v Baltica. Ten samples were collected from the Vistula River estuary (Fig. 1).

At each sampling event, aliquots from three depths of 0, 2.5, and 5 m were mixed

Fig. 1. Locations of sampling sites in the Vistula River estuary (Baltic Sea). The study was performed between June 16

and June 24

, 2005

together and treated as a sample representative for the surface waters. These mixed samples were subsequently fixed with glutaraldehyde for the analysis of small het- erotrophic nanoflagellates (up to 5 µm in size), and with acid Lugol’s solution for the analysis of larger heterotrophic nanoflagellates (5-20 µm in size), dinoflagel- lates, and ciliates. Sampling was accompanied by simultaneous measurements of water temperature and salinity. At a number of stations during the cruise, the concen- tration of chlorophyll a, phytoplankton biomass, primary production, bacterial bio- mass, and bacterial secondary production were also measured (see Wielgat-Rychert et al. 2013).

The smallest heterotrophic nanoflagellates (up to 5 µm in size) were studied in sam-

ples fixed with glutaraldehyde (0.5% final concentration). The nanoflagellates were

concentrated onto nucleopore filters, stained with primulin, and counted under an

epifluorescence microscope (Caron 1983). Only heterotrophic organisms, i.e., those

without chlorophyll autofluorescence, were counted. Their biomass was estimated

from cell volumes with the coefficient of 0.22 pgC µm

(Børsheim and Bratbak 1987).

Larger heterotrophic nanoflagellates (5-20 µm in size) were analyzed together with heterotrophic dinoflagellates and ciliates. The analyses were performed under an in- verted microscope (Utermöhl’s sedimentation technique, HELCOM 2001) in sam- ples fixed with acid Lugol’s solution (0.5% final concentration). Specimens were identified to the most accurate taxonomic level possible. Heterotrophic nanoflagel- lates were identified according to Thomsen (1992), heterotrophic dinoflagellates ac- cording to Drebes (1974), Thomsen (1992), Identyfying marine... (1997), and Throndsen et al. (2007), and ciliates according to Marshall (1969), Witek (1994), Maeda and Carey (1985), and a web-based guide (Strüder-Kypke and Montagnes 2002). We used taxonomic names that were accepted in the World Register of Ma- rine Species (WoRMS 2014). The biomasses (CC, carbon content, pgC) of hetero- trophic nanoflagellates, dinoflagellates, and naked ciliates were calculated from cell volumes (CV, µm

) using the following allometric formula (Menden-Deuer and Lessard 2000):

CC = 0.216 × CV

The biomass of tintinnid ciliates was calculated from the volume of lorica (LV, µm

) according to Verity and Langdon (1984):

CC = 0.053 × LV + 444.5

As was mentioned in the Introduction, some protozoa are mixotrophic. In this study, only obligatory heterotrophic nanoflagellates and dinoflagellates were counted; their nutritional modes were identified according to the guides mentioned above and a list published by Olenina et al. (2006). In the case of ciliates, all cells except Meso- dinium rubrum Lohmann were counted. M. rubrum was excluded because of its pre- vailing autotrophy (Esteban et al. 2010). Since other mixotrophic ciliates could not be identified in the Lugol-fixed samples, most probably some mixotrophic ciliates were classified as heterotrophic forms.

Considerable amounts of the flagellate Ebria tripartita (Schumann) Lemmermann were observed among the protozoa. This organism was grouped together with het- erotrophic dinoflagellates because of its similar size range and food preferences (dia- toms, Drebes 1974, Hargraves 2002), as was done by Bralewska and Witek (1995).

RESULTS

The surface water temperature at sampling sites varied between 16.5ºC (E52a, Fig.

1) and 13.5ºC (P63), and the salinity between 4.4‰ (E52a, 3.2 nM from the mouth of the Vistula River) and 7.2‰ (P63, offshore station). The last value was compara- ble to the average salinity observed in the surface waters of the southern Baltic Sea (~7.5‰, Majewski 1987).

Only the larger forms in the heterotrophic nanoflagellate communities (larger than

5 µm) could be identified taxonomically, i.e. those observed under an inverted mi-

croscope. The most frequently observed were Kathablepharis remigera (Vørs) Clay

& Krugens, Leucocryptos marina (Braarud) Butcher, and Telonema sp. They were particularly abundant at station G2 situated in the Gdańsk Deep (Fig. 1). Close to the river mouth their biomass was lower, and nanoflagellate community was dominated by organisms that were not identified taxonomically.

As was mentioned above, Ebria tripartita was grouped together with the dinofla- gellates. E. tripartita was observed at all stations and contributed from 19% to 84%

(mean 46%) of the combined biomass of E. tripartita and dinoflagellates. Other common organisms in this group were thin-walled dinoflagellates belonging to Peridiniales (10-20 µm) and naked forms belonging to Gymnodiniales (10-30 µm).

The former contributed from 30% to 50% of the dinoflagellate biomass (including E. tripartita) at almost all the stations studied; however, thin-walled Peridiniales were absent at offshore station P63 and station E52a located close to the Vistula River mouth. Gymnodiniales non def. were observed at all the stations studied with contributions to dinoflagellate biomass ranging from 5% to 29%. At the offshore site, unidentified Gymnodiniales were accompanied by Gyrodinium fusiforme Kofoid

& Swezy, G. spirale (Bergh) Kofoid & Swezy, and Katodinium glaucum (Lebour) Loeblich III. The thecate dinoflagellates: Phalacroma rotundatum (Claparéde & Lach- mann) Kofoid & Michener, Protoperidinium bipes (Paulsen) Balech, P. brevipes (Paulsen) Balech, and Oblea rotunda (Lebour) Balech ex Sournia, and the thick- walled Peridiniales non def. (10-30 µm) contributed only 7% of the dinoflagellate biomass, on average, and only at one station (E83) did their contribution reach 21%.

Among the ciliates, the most frequently observed genera were Strombidium, Pelago- strobilidium, Rimostrombidium, Tintinnopsis, Askenasia, Urotricha, Euplotes, Vorti- cella, Mesodinium (heterotrophic species), and Balanion comatum Wulff, while Lohmanniella oviformis Leegaard and hypotrichs other than Euplotes sp. were less frequent. At station E52a, located only 3.2 nM from the mouth of the Vistula River, the majority of the ciliate biomass (59%) was contributed by tintinnids (Tintinnopsis parvula Jörgensen, T. lobiancoi Daday, T. beroidea Stein). Their contribution gradu- ally decreased with increasing distance from the river mouth (stations: E83, E53, and E90; Fig. 1), and tintinnids were generally absent at stations located 11.5 nM (station E60) or more from the river mouth. This trend was statistically significant (p = 0.04). Distinct abundances of Euplotes sp., Vorticella spp., and the freshwater ciliate Phascolodon sp. were also observed close to the river mouth.

The combined biomass of protozoa was up to 162 µgC l

close to the river mouth and gradually decreased to 20.7 µgC l

at the most offshore station P63 located 64.3 nM from the mouth (Fig. 2). The negative correlation between distance from the mouth and protozoan biomass was highly statistically significant (p = 0.002), whereas the corre- sponding relation between water salinity (indicating an impact of fresh water) and pro- tozoan biomass was non-significant (Table 1). Heterotrophic dinoflagellates and E.

tripartita contributed the majority of the protozoan biomass; therefore, the negative cor-

relation between distance from the river mouth and their biomass estimates resembled

that for the entire community (Table 1). Ciliate biomass was not significantly correlated

with salinity or distance from the mouth, mainly because of their high biomass recorded

at the most offshore station P63. It is noteworthy that heterotrophic nanoflagellates dis-

played a strong negative correlation with water salinity (Table 1).

Fig. 2. Protozoan biomass decreased with increasing dis- tance from the mouth of the Vistula River. The negative correlation is highly statistically significant (p = 0.002)

Table 1 Correlation coefficients between (i) protozoan biomass and (ii) water salinity

and distance from the Vistula River mouth. Only statistically significant coefficients (p < 0.05) are depicted; non-significant ones are indicated as n.s.

Protozoan biomasses were log-transformed prior to analysis

Protozoan biomass Salinity Distance

All protozoa n.s. -0.85

Heterotrophic ciliates n.s. n.s.

Heterotrophic dinoflagellates (incl. E. tripartita) n.s. -0.83

Ebria tripartita only n.s. -0.75

Heterotrophic nanoflagellates -0.78 n.s.

The protozoan community composition differed between offshore station P63 and

the remaining stations located closer to the river mouth (Fig. 3). Heterotrophic

nanoflagellates, dinoflagellates, and ciliates contributed 57%, 25% and 18%, respec-

tively, to community biomass at station P63. At the remaining stations, heterotrophic

dinoflagellates were highly significant (Fig. 3) since they contributed 71% of the

Fig. 3. Composition of protozoan communities observed in the Vistula River estuary in June 2005. Ebria tripartita was grouped together with heterotrophic dinoflagellates

protozoan biomass on average (standard deviation, SD = 13%), while heterotrophic nanoflagellates contributed 22% (SD = 10%), and ciliates only 7% (SD = 8%). The differences between offshore station P63 and the other stations are statistically sig- nificant (the three-sigma rule).

DISCUSSION

Taxonomic composition and biomass

The taxa noted during the present study were reported previously in the Baltic Sea

(Smetacek 1981, Mackiewicz 1991, Bralewska and Witek 1995, Witek 1998, Setälä

and Kivi 2003, Witek and Pliński 2005, Olenina et al. 2006, Mironova et al. 2009,

Grinienė et al. 2011, Rychert 2011). Elevated abundances of Ebria tripartita and

tintinnid ciliates were observed close to the river mouth. Similar observations were

also reported from other estuaries for both E. tripartita (Pollehne et al. 1995,

Wrzesińska-Kwiecień and Mackiewicz 1995) and tintinnids (Revelante and Gilmartin

1987, Sanders 1987, Leakey et al. 1993, Wrzesińska-Kwiecień and Mackiewicz 1995,

Urrutxurtu et al. 2003, but see Christaki et al. 2009). E. tripartita feeds on diatoms

(Drebes 1974, Hargraves 2002), and analyses of phytoplankton communities (the

same samples, Wielgat-Rychert et al. 2013) indeed demonstrated the dominance of

centric diatoms among the algae in riverine waters and close to the river mouth. To-

wards the open sea, diatoms were gradually replaced by autotrophic dinoflagellates

and cyanobacteria. Therefore, E. tripartita occurrence was governed by food re-

sources. Tintinnids feed on many groups of algae and protozoa, and their preferred

food includes nanoplanktonic dinoflagellates, nanoflagellates, and pico/nanoplankto- nic cyanobacteria (Sanders 1987, Urrutxurtu et al. 2003, Montagnes 2013), whereas diatoms are rarely ingested. Elevated tintinnid abundances in the estuary seemed to be coupled simply with higher food concentrations; however, Sanders (1987) and Urrutxurtu et al. (2003) also discussed the tintinnid preference for higher water tem- peratures. Few freshwater species (e.g., ciliate Phascolodon sp.) were noted close to the river mouth however, protozoan communities in the salinity gradient of 4.4-7.2‰

and at a distance of 3.2-64.3 nM from the mouth comprised marine protozoa.

The protozoan biomass corresponded well with values observed previously in the Baltic Sea during early summer (June). Biomass of heterotrophic nanoflagellates ranged from 12 to 36 µgC l

and was comparable to the value observed in the coastal zone of the Gulf of Gdańsk in June 2003 (29 µgC l

, Rychert et al. 2013).

The biomass of heterotrophic dinoflagellates (including E. tripartita) ranged from 5 to 130 µgC l

, which was similar to values observed in June in the coastal zone of the Gulf of Gdańsk: (~40 µgC l

, Kwiatkowska 1999), those reported from the inner part of the Gulf of Gdańsk (30-50 µgC l

, Bralewska and Witek 1995), and those noted in offshore waters in June 2006 and June 2007 (2.5-16 µgC l

; Kownacka, personal communication). The ciliate biomass (1-17 µgC l

) was far lower than that observed in June in the coastal zone of the Gulf of Gdańsk (71 µgC l

, Rychert et al.

2013) and was comparable to that observed in June in the inner waters of the Gulf of Gdańsk (~10 µgC l

, Witek 1998), in offshore waters in June 2006 and June 2007 (3-9 µgC l

; Kownacka, personal communication), and also in the Neva Estuary (Baltic Sea, 5-11 µgC l

, Mironova et al. 2012).

Food resources for protozoa

In estuaries the protozoan biomass decreases towards the open sea (Revelante and Gilmartin 1987, McManus and Fuhrman 1990, Arndt 1991, Christaki et al. 2009). In the present study a five-fold decrease was observed (Fig. 2). Phytoplankton biomass and chlorophyll a concentrations studied simultaneously (Wielgat-Rychert et al.

2013) demonstrated comparable decreases that were related to water salinity, which indicated conservative mixing, or dilution, of river waters in sea waters. At station E52a, 3.2 nM from the river mouth, chlorophyll a concentration amounted to 32.9 mg m

, corresponding to phytoplankton biomass of 867 µgC l

, and it gradual- ly decreased to 2.9-3.5 mg m

at the most offshore stations (phytoplankton biomass:

132-341 µgC l

). For more data about phytoplankton, studied at higher number of sampling stations, see Wielgat-Rychert et al. (2013). On the contrary, bacterial bio- mass dropped abruptly near the river mouth, while further decreases were less steep (Wielgat-Rychert et al. 2013). That initial drop in bacterial abundance and biomass occurred between the river mouth and the closest station at which protozoan com- munities were studied (E52a, 3.2 nM from the mouth). At stations where protozoan communities were studied bacterial abundance and biomass amounted to 1.62-2.26

× 10

l

and 22.5-42.9 µgC l

, respectively. For more data see Wielgat-Rychert et al.

(2013).

Protozoan biomass corresponded to an average of 21% of the phytoplankton bio-

mass (range 12-30%), and it exceeded bacterial biomass by an average of three

times (range: 0.5-4.6). These data on phytoplankton and bacterioplankton are from Wielgat-Rychert et al. (2013). Correlation analysis performed with log-transformed data demonstrated that the combined protozoan biomass and that of particular proto- zoan groups depended on phytoplankton biomass, chlorophyll a concentration, or primary production (Table 2), but, with the exception of heterotrophic ciliates, not on bacterial biomass or bacterial secondary production (Table 2). Dependencies be- tween (i) phytoplankton and (ii) heterotrophic ciliates or dinoflagellates are typical

Table 2 Correlation coefficients between (i) protozoan biomass and (ii) phytoplankton and bacterial parameters. Only statistically significant coefficients (p < 0.05) are depicted; non-significant ones are indicated as n.s. Data were log-transformed prior to analysis

Protozoan biomass Phytopl.

biomass

Chloro- phyll a

Primary production

Bacterial biomass

Bacterial production

All protozoa 0.81 n.s. 0.74 n.s. n.s.

Het. ciliates n.s. 0.76 n.s. n.s. 0.68

Het. dinoflagellates

(incl. E. tripartita) 0.75 n.s. 0.73 n.s. n.s.

Ebria tripartita only 0.82 n.s. 0.66 n.s. n.s.

Het. nanoflagellates 0.73 0.64 n.s. n.s. n.s.

(Hansen 1991, Rollwagen-Bollens et al. 2011). Conversely, a strong correlation be- tween phytoplankton biomass (or chlorophyll a) and heterotrophic nanoflagellates was rather unusual and indicated that the latter fed on small algae. Cyanobacteria and autotrophic flagellates were observed in the samples (Wielgat-Rychert et al.

2013), but in rather small numbers. However, that nanoflagellate grazing on small algae was also suggested by the high fraction of nanoflagellates exceeding 5 µm, be- cause heterotrophic nanoflagellates smaller than 5 µm are generally bacterivorous, whereas larger ones feed on cyanobacteria and both heterotrophic and autotrophic flagellates (Sherr and Sherr 1991, Brandt and Sleigh 2000, Sherr and Sherr 2002, Piwosz and Pernthaler 2010). In the current study, the heterotrophic nanoflagellate biomass was evenly distributed between fractions < 5 µm and > 5 µm, whereas other authors (McManus and Fuhrman 1990, Brandt and Sleigh 2000 and references there- in, Calbet et al. 2001, Christaki et al. 2009) reported that in marine environments the majority of the biomass, and often almost the entire biomass, is typically contributed by the smallest bacterivorous nanoflagellates.

In the Introduction we posed the question of whether the availability of phytoplank- ton or bacteria was primarily responsible for shaping the composition of protozoan communities along the Vistula River estuary. Correlation analysis and the simulta- neous study of bacterial parameters (Table 2) precluded the significant impact of bacteria on protozoan communities within a distance from the river mouth studied (3.2-64.3 nM). Contrarily, protozoan biomass, especially the dominant biomass of E.

tripartita and heterotrophic dinoflagellates, was correlated with chlorophyll a, phy-

toplankton biomass, or primary production. Among the ciliates, which were the only group significantly correlated with bacterial production (Table 2), typical bacterivo- rous organisms, e.g., scuticociliates, were not observed. Consequently, bacteria must have been grazed by omnivorous ciliates that also fed on algae, e.g., oligotrichs and choreotrichs. It should be emphasized that ciliate biomass was correlated with bacte- rial production but not with bacterial biomass. This is because bacterial production is a better parameter for these types of analyses since only a fraction of bacterial cells are metabolically active and grow (Vaqué et al. 2001). Compared directly within the euphotic zone, bacterial secondary production corresponded to an average of 6% to 25% of phytoplankton primary production (Wielgat-Rychert et al. 2013), which was within typical ranges (Cole et al. 1988). This means that phytoplankton primary pro- duction was almost one order of magnitude higher than bacterial secondary produc- tion. However, in fact, the discrepancy between protozoan food of phytoplankton and bacterial origin was lower, because only a fraction of phytoplankton primary production that is contributed by small algae is consumed by protozoa, and the re- maining part is grazed directly by rotifers and crustaceans (Azam et al. 1983, Landry and Kirchman 2002, Sherr and Sherr 2002). In conclusion, phytoplankton produc- tion exerted a stronger impact on the protozoan community than did bacterial pro- duction.

Heterotrophic dinoflagellates in the Gulf of Gdańsk

In marine environments biomasses contributed by heterotrophic nanoflagellates, dinoflagellates, and ciliates are comparable (Arndt 1991, Hansen 1991, Sherr and Sherr 2002, Verity and Borkman 2010, Rychert et al. 2013). In the present study, with the exception of offshore station P63, protozoan communities were atypically dominated by heterotrophic dinoflagellates (71% of the biomass on average, includ- ing E. tripartita). It confirmed the significance of dinoflagellates in the Gulf of Gdańsk that was reported previously by Bralewska and Witek (1995) who observed that within the gulf the biomass of heterotrophic dinoflagellates was twice as high as ciliate biomass, whereas outside of it the biomass of heterotrophic dinoflagellates and ciliates was balanced. Another study (Rychert 2011) demonstrated that in June heterotrophic dinoflagellates contributed only 29-39% to the protozoan biomass in the well-oxygenated, near-bottom zone of the Gulf of Gdańsk, which is less influ- enced by riverine fresh water. Similarly, at a sampling site in Sopot, which is located in the coastal zone of the Gulf of Gdańsk but is distant from the river mouth, hetero- trophic dinoflagellates contributed only 30% of protozoan biomass on an annual ba- sis with similar values recorded in June (Rychert et al. 2013). Consequently, the high abundances of heterotrophic dinoflagellates, observed in this study seemed to be re- lated to the inflow of fresh water from the Vistula River. Heterotrophic dinoflagel- lates and E. tripartita were not delivered by the river, but they must have grown in the estuary, because heterotrophic dinoflagellates were not noted in the riverine wa- ter and the abundance of E. tripartita was far lower than that observed in the estuary (data not shown).

This study included the three main protozoan groups of heterotrophic nanoflagel-

lates, dinoflagellates, and ciliates. Fourth group, amoebae in pelagic marine waters

has long been considered insignificant (Caron and Swanberg 1990), but some stud- ies underscored their importance in estuaries (Sherr and Sherr 2002, Rogerson et al.

2003, Lesen et al. 2010). Further investigations on protozoan communities in estuar- ies should therefore take amoebae into account.

CONCLUSIONS

1. Along the estuary, in the surface waters a five-fold decrease in protozoan biomass was observed. The negative correlation between distance from the mouth and protozoan biomass was highly statistically significant.

2. Within the estuary, the majority of the biomass was contributed by heterotrophic dinoflagellates and Ebria tripartita (71% on average), whereas at the offshore station, the contribution of heterotrophic dinoflagellates decreased to a typical value of 25%. The differences between offshore station and stations located within the estuary are statistically significant (the three-sigma rule). The study confirmed high significance of heterotrophic dinoflagellates and E. tripartita in the Gulf of Gdańsk.

3. Correlation analysis revealed that phytoplankton production exerted a stronger impact on the composition of the protozoan community than did bacterial production.

The study was supported by the Polish Ministry of Science and Higher Education as statutory activities of Department of Ecology of the Pomeranian University in Słupsk (project 11.6.13) and statutory activities of the Department of Fisheries Oceanography and Marine Ecology of the National Marine Fisheries Research In- stitute (projects O-147 and P2-1).

REFERENCES

Arndt H., 1991. On the importance of planktonic protozoans in the eutrophication process of the Baltic Sea. Int. Revue ges. Hydrobiol., 3, 387-396.

Azam F., Fenchel T., Field J.D., Gray J.S., Meyer-Reil L.A., Thingstad F., 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser., 10, 257-263.

Beaver J.R., Crisman T.L., 1982. The trophic response of ciliated protozoans in freshwater lakes. Limnol. Oceanogr., 27, 246-253.

Børsheim K.Y., Bratbak G., 1987. Cell volume to carbon conversion factors for a bacterio- vorous Monas sp. enriched from seawater. Mar. Ecol. Prog. Ser., 36, 171-175.

Bralewska J., Witek Z., 1995. Heterotrophic dinoflagellates in the ecosystem of the Gulf of Gdańsk. Mar. Ecol. Prog. Ser., 117, 241-248.

Brandt S.M., Sleigh M.A., 2000. The quantitative occurrence of different taxa of hetero- trophic flagellates in Southampton Water, U.K. Estuar. Coast. Shelf Sci., 51, 91-102.

Calbet A., Landry M.R., Nunnery S., 2001. Bacteria-flagellate interactions in the microbial food web of the oligotrophic subtropical North Pacific. Aquat. Microb. Ecol., 23, 283-292.

Caron D.A., 1983. Technique for enumeration of heterotrophic and phototrophic nano- plankton, using epifluorescence microscopy, and comparison with other procedures. Appl.

Environ. Microbiol., 46, 491-498.

Caron D.A., Swanberg N.R., 1990. The ecology of planktonic sarcodines. Aquat. Sci., 3, 147- 180.

Christaki U., Courties C., Joux F., Jeffrey W.H., Neveux J., Naudin J.-J., 2009. Community structure and trophic role of ciliates and heterotrophic nanoflagellates in Rhone River diluted mesoscale structures (NW Mediterranean Sea). Aquat. Microb. Ecol., 57, 263- 277.

Cole J.J., Findlay S., Pace M.L., 1988. Bacterial production in fresh and saltwater ecosystems:

a cross-system overview. Mar. Ecol. Prog. Ser., 43, 1-10.

Drebes G., 1974. Marines Phytoplankton, Georg Thieme Verlag, Stuttgart, (in German).

Ducklow H.W., Purdie D.A., Williams P.J. le B., Davies J.M., 1986. Bacterioplankton: a sink for carbon in a coastal marine plankton community. Science, 232, 865-867.

Dutz J., Peters J., 2008. Importance and nutritional value of large ciliates for the reproduction of Acartia clausi during the post spring-bloom period in the North Sea. Aquat. Microb.

Ecol., 50, 261-277.

Esteban G.F., Fenchel T., Finlay B.J., 2010. Mixotrophy in ciliates. Protist, 161, 621-641.

Fenchel T., 1982. Ecology of heterotrophic microflagellates. IV. Quantitative occurrence and importance as bacterial consumers. Mar. Ecol. Prog. Ser., 9, 35-42.

Grinienė E., Mažeikaitė S., Gasiūnaitė Z.R., 2011. Inventory of the taxonomical composition of the plankton ciliates in the Curonian Lagoon (SE Baltic Sea). Oceanol. Hydrobiol.

Stud., 40, 86-95.

Hansen P.J., 1991. Quantitative importance and trophic role of heterotrophic dinoflagellates in a coastal pelagial food web. Mar. Ecol. Prog. Ser., 73, 253-261.

Hargraves P.E., 2002. The ebridian flagellates Ebria and Hermesinum. Plankton Biol. Ecol., 49, 9-16.

HELCOM, 2001. Manual for marine monitoring in the COMBINE programme of HELCOM, www.helcom.fi/groups/monas/CombineManual/AnnexesC/en_GB/annex6/

HELCOM, 2004, The fourth Baltic Sea pollution load compilation (PLC-4), Balt. Sea Envi- ron. Proc., 93.

Identifying marine phytoplankton. 1997. (Ed.) C.R. Tomas, Academic Press, San Diego.

Jeong H.J., 1999. The ecological roles of heterotrophic dinoflagellates in marine planktonic community. J. Eukaryot. Microbiol., 46, 390-396.

Kwiatkowska M., 1999. Autotroficzne i heterotroficzne bruzdnice w przybrzeżnej strefie Zatoki Gdańskiej. (Autotrophic and heterotrophic dinoflagellates in the coastal zone of the Gulf of Gdańsk). M.Sc. Thesis, University of Gdańsk, Gdańsk, (in Polish).

Landry M.R., Kirchman D.L., 2002. Microbial community structure and variability in the tropical Pacific. Deep-Sea Res. II, 49, 2669-2693.

Leakey R.J.G., Burkill P.H., Sleigh M.A., 1993. Planktonic ciliates in Southampton water:

quantitative taxonomic studies. J. Mar. Biol. Ass. U.K., 73, 579-594.

Lesen A.E., Juhl A.R., Anderson O.R., 2010. Heterotrophic microplankton in the lower Hudson River Estuary: potential importance of naked, planktonic amebas for bacterivory and carbon flux. Aquat. Microb. Ecol., 61, 45-56.

Mackiewicz T., 1991. Composition and seasonal changes of nanoflagellates in the Gdańsk Basin (Southern Baltic). Acta Ichthyol. Piscat., 21 Suppl., 125-134.

Maeda M., Carey P.G., 1985. An illustrated guide to the species of the family Strombidiidae (Oligotrichida), free swimming protozoa common in aquatic environment. Bull. Ocean.

Res. Inst. Univ. Tokyo, 19, 1-68.

Majewski A., 1987. Charakterystyka wód. W: Bałtyk Południowy. (Characteristics of waters. In:

The Southern Baltic). (Ed.) B. Augustowski, Ossolineum, Wrocław, 173-217, (in Polish).

Marshall S.M., 1969. Protozoa. Order: Tintinnida. In: Fishes d’identification du Zooplancton.

(Eds) J.H. Fraser and V.K. Hanson, Cons. Int. Explor. Mer. Zooplankton Sheets, Charlot-

tenlund, 117-127.

McManus G.B., Fuhrman J.A., 1990. Mesoscale and seasonal variability of heterotrophic nanoflagellate abundance in an estuarine outflow plume. Mar. Ecol. Prog. Ser., 61, 207- 213.

Menden-Deuer S., Lessard E., 2000. Carbon to volume relationships for dinoflagellates, dia- toms, and other protist plankton. Limnol. Oceanogr., 45, 569-579.

Mironova E.I., Telesh I.V., Skarlato S.O., 2009. Planktonic ciliates of the Baltic Sea (a review).

Inland Water Biol., 2, 13-24.

Mironova E., Telesh I., Skarlato S., 2012. Diversity and seasonality in structure of ciliate communities in the Neva Estuary (Baltic Sea). J. Plankton Res., 34, 208-220.

Montagnes D.J.S., 2013. Ecophysiology and behaviour of tintinnids. In: The biology and ecology of tintinnid ciliates. Models for marine plankton. (Eds) J.R. Dolan, D.J.S. Mon- tagnes, S. Agatha, D.W. Coats, D.K. Stoecker, Wiley-Blackwell, Chichester, 85-121.

Montagnes D.J.S., Kimmance S.A., Tsounis G., Gumbs J.C., 2001. Combined effect of tem- perature and food concentration on the grazing rate of the rotifer Brachionus plicatilis.

Mar. Biol., 139, 975-979.

Olenina I., Hajdu S., Edler L., Andersson A., Wasmund N., Busch S., Gobel J., Gromisz S., Huseby S., Huttunen M., Jaanus A., Kekkonen P., Ledaine I., Ziemkiewicz E., 2006. Bio- volumes and size-classes of phytoplankton in the Baltic Sea, HELCOM Baltic Sea Envi- ronment Proceedings, 106.

Piwosz K., Pernthaler J., 2010. Seasonal population dynamics and trophic role of planktonic nanoflagellates in coastal surface waters of the Southern Baltic Sea. Environ. Microbiol., 12, 364-377.

Pollehne F., Busch S., Jost G., Meyer-Harms B., Nausch M., Reckermann M., Schaening P., Setzkorn D., Wasmund N., Witek Z., 1995. Primary production patterns and heterotrophic use of organic material in the Pomeranian Bay (southern Baltic). Bull. Sea Fish. Inst., 136, 43-60.

Revelante N., Gilmartin M., 1987. Seasonal cycle of the ciliated protozoan and micrometa- zoan biomass in a Gulf of Maine Estuary. Estuar. Coast. Shelf Sci., 25, 581-598.

Rogerson A., Anderson O.R., Vogel C., 2003. Are planktonic naked amoebae predominately floc associated or free in the water column? J. Plankton Res., 25, 1359-1365.

Rollwagen-Bollens G., Gifford S., Bollens S.M., 2011. The role of protistan microzooplank- ton in the upper San Francisco Estuary planktonic food web: source or sink? Estuar.

Coast., 34, 1026-1038.

Rychert K., 2011. Communities of heterotrophic protists (protozoa) in the near-bottom zone of the Gdańsk Basin. Oceanol. Hydrobiol. Stud., 40, 68-73.

Rychert K., Spich K., Laskus K., Pączkowska M., Wielgat-Rychert M., Sojda G., 2013. Com- position of protozoan communities at two stations in the coastal zone of the southern Bal- tic Sea. Oceanol. Hydrobiol. Stud., 42, 268-276.

Sanders R.W., 1987. Tintinnids and other microzooplankton – seasonal distributions and rela- tionship to resources and hydrography in a Main estuary. J. Plankton Res., 9, 65-77.

Setälä O., Kivi K., 2003. Planktonic ciliates in the Baltic Sea in summer: distribution, species association and estimated grazing impact. Aquat. Microb. Ecol., 32, 287-297.

Sherr B.F., Sherr E.B., 1991. Proportional distribution of total numbers, biovolume, and bac- terivory among size classes of 2-20 µm nonpigmented marine flagellates. Mar. Microb.

Food Webs, 5, 227-237.

Sherr E.B., Sherr B.F., 2002. Significance of predation by protists in aquatic microbial food webs. Antonie Leeuwenhoek, 81, 293-308.

Smetacek V., 1981. The annual cycle of protozooplankton in the Kiel Bight. Mar. Biol., 63, 1-11.

Strom S.L., Postel J.R., Booth B.C., 1993. Abundance, variability, and potential grazing im-

pact of planktonic ciliates in the open subarctic Pacific Ocean. Prog. Oceanog., 32, 185-

203.

Strüder-Kypke M.C., Montagnes D.J.S., 2002. Development of web-based guides to planktonic protists. Aquat. Microb. Ecol., 27, 203-207.

Thomsen H.A., 1992. Plankton i de indre danske forvande. Analyse af forekomsten af alger og heterotrofe protister (ekskl.ciliater) i Kattegat. (Plankton from inner Danish waters. An analysis of the autotrophic and heterotrophic protists (excl. ciliates) in Kattegat).

Havforskning fra Miløstyrelsen, 11, (in Danish).

Throndsen J., Hasle G.R., Tangen K., 2007. Phytoplankton of Norwegian coastal waters.

Almater Forlag As, Oslo.

Urrutxurtu I., Orive E., de la Sota A., 2003. Seasonal dynamics of ciliated protozoa and their potential food in an eutrophic estuary (Bay of Biscay). Est. Coast. Shelf Sci., 57, 1169- 1182.

Vaqué D., Casamayor E.O., Gasol J.M., 2001. Dynamics of whole community bacterial pro- duction and grazing losses in seawater incubations as related to the changes in the propor- tions of bacteria with different DNA content. Aquat. Microb. Ecol., 25, 163-177.

Verity P.G., Borkman D.G., 2010. A decade of change in the Skidaway River Estuary. III.

Plankton. Estuar. Coast., 33, 513-540.

Verity P.G., Langdon C., 1984. Relationships between lorica volume, carbon, nitrogen, and ATP content of tintinnids in Narragansett Bay. J. Plankton Res., 6, 859-867.

Wielgat-Rychert M., Ameryk A., Jarosiewicz A., Kownacka J., Rychert K., Szymanek L., Za- lewski M., Agatova A., Lapina N., Torgunova N., 2013. Impact of the inflow of the Vis- tula River waters on the pelagic zone in the Gulf of Gdańsk. Oceanologia, 55, 859-886.

Witek M., 1994. Planktonowe orzęski Basenu Gdańskiego. (Planktonic ciliates of the Gdańsk Basin). Ph.D. Thesis, Sea Fisheries Institute, Gdynia, (in Polish).

Witek M., 1998. Annual changes of abundance and biomass of planktonic ciliates in the Gdańsk Basin, southern Baltic. Internat. Rev. Hydrobiol., 83, 163-182.

Witek B., Pliński M., 2005. The occurrence of dinoflagellates in the phytoplankton of the Gulf of Gdańsk coastal zone in 1994-1997. Oceanol. Hydrobiol. Stud., 2, 63-70.

WoRMS Editorial Board, 2014. World Register of Marine Species, www.marinespecies.org Wrzesińska-Kwiecień M., Mackiewicz T., 1995. Protozooplankton of the Pomeranian Bay

(southern Balic). Bull. Sea Fish. Inst., 136, 89-95.

ZBIOROWISKA PIERWOTNIAKÓW W ESTUARIUM WISŁY (MORZE BAŁTYCKIE)

Streszczenie

Zbiorowiska pierwotniaków (heterotroficzne wiciowce, bruzdnice i orzęski) badano wzdłuż estuarium rzeki Wisły (Bałtyk Południowy). Badania przeprowadzono po wiosennym zakwicie fitoplanktonu, pobierając próby z wód powierzchniowych. Biomasa pierwotniaków wynosiła 64,1-162 µgC l

blisko ujścia rzeki i stopniowo spadała do 20,7 µgC l

na najbardziej wysu- niętym w morze stanowisku (64,3 mil morskich od ujścia rzeki). Zaobserwowana zależność pomiędzy odległością od ujścia a biomasą pierwotniaków była wysoce istotna statystycznie.

W obrębie estuarium większość biomasy zbiorowiska pierwotniaków (średnio 71%) stanowi-

ły heterotroficzne bruzdnice (razem z Ebria tripartita), podczas gdy heterotroficzne wiciow-

ce i orzęski osiągały odpowiednio 22% i 7% całkowitej biomasy pierwotniaków. Na najbar-

dziej wysuniętym w morze stanowisku udział heterotroficznych bruzdnic do biomasy spadał

do 25%, co jest wartością typową, ponieważ w wodach morskich trzy wymienione grupy

pierwotniaków zwykle wykazują porównywalną biomasę. Badania potwierdziły szczególnie

duże znaczenie heterotroficznych bruzdnic i Ebria tripartita w Zatoce Gdańskiej i wykazały,

że ma to związek z napływem wód wnoszonych przez Wisłę. Analiza statystyczna wykazała,

że wszystkie grupy pierwotniaków były powiązane z biomasą fitoplanktonu lub wielkością

produkcji pierwotnej, podczas gdy tylko heterotroficzne orzęski wykazały istotną korelację

z wielkością produkcji wtórnej bakterioplanktonu.