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AQUATIC MICROBIAL ECOLOGY Vol. 11: 161-169,1996 Published November 7 Aquat Microb Ecol

Sporadic high abundances of naked amoebae in Black Sea plankton

Sergey A. Murzovl,David A. caron21*

'Institute of Biology of the Southern Seas, Ukrainian Academy of Sciences, 2 Nakhimov Ave., Sevastopol335011, Crimea, Ukraine 2~iologyDepartment. Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543. USA

ABSTRACT: The abundance of non-testate ('naked'), free-living amoebae was examined in surface waters of the Black Sea during 5 cruises in 1992 and 1993. Amoebae (520pm in size) were observed in samples collected during 2 of the 5 cruises at abundances up to approximately 380 ml-l The abun- dance and biomass of amoebae were low relative to other nanoplanktonic when averaged over all stations, but amoebae occasionally constituted a major component of the heterotrophic nanoplanktonic assemblage (up to 16% of abundance and 93% of biomass of the protozoa 2 to 20 pm in size). Abundances of amoebae correlated weakly with the total number of heterotrophic nanoplank- ton, and with ash free dry weight, but not with several other physical, chemical and biological parame- ters. Based on the highest abundances of naked amoebae observed in this study we suggest that these protozoa can occasionally be important in energy and elemental cycling in Black Sea plankton.

KEYWORDS: Amoebae . Protozoa . Plankton

INTRODUCTION There is a general acceptance that amoebae play a significant role in nutrient cycling within soil ecosys- Recent synopses of research on the 'microbial loop' tems (Alexander 1961). There is also a perception that (sensu latu Pomeroy 1974, Azam et al. 1983) indicate they are of minor importance in most aquatic environ- that there is a growing consensus among ecologists on ments (particularly in the plankton) where many of the major features of aquatic microbial food and dominate the biomass and activities of webs (Hobbie 1994). In particular, major roles have protozoan assemblages (Fenchel 1987, Capriulo 1990, been firmly established for flagellated and ciliated pro- Reid et al. 1991). While this perception may be true on tozoa (Sherr & Sherr 1994). Nevertheless, there are still average, our knowledge of the ecology of free-living a number of aspects of protozoan assemblages and amoebae is still too rudimentary to place firm limits on their interactions which remain enigmatic. The impor- the degree to which they contribute to living biomass tance of free-living, non- (the 'naked' or affect energy transfer in planktonic ecosystems. amoebae) in the energetics of aquatic communities is Difficulties associated with counting and identifying clearly one of these features. The benchmark mono- amoebae in nature hinder our understanding of their graph of Schaeffer (1926) established the presence of a ecology. Species diversity of the non-testate amoebae diverse fauna of amoebae in aquatic ecosystems. Since is still relatively poorly characterized because identifi- that study, however, there has been much less infor- cations are based in part on features of the living cells. mation acquired on the abundances and interactions of In addition, the trophic activities of many of the species this assemblage in aquatic food webs than for other are inadequately defined, making it difficult to esti- components of protozoan communities. mate the contribution of amoebae to energy and ele- mental flow in natural aquatic communities. Given these limitations, it is not surprising that much of the 'Addressee for reprint requests. E-mail: [email protected] interest in free-living amoebae has been limited to

O Inter-Research 1996 Aquat Microb Ecol 11: 161-169, 1996

potential human pathogens (Sawyer et al. 1977, 1993, air/water interface (Davis 1976, Sieburth et al. 1976, Daggett et al. 1982, Kyle & Noblet 1985, Sawyer 1989). Davis et al. 1978, Kyle & Noblet 1987), on the surfaces Amoebae are widely distributed in both freshwater of microalgae or macroalgae (Cook et al. 1974, Ander- and marine plankton communities, although there are son 1977, Kyle & Noblet 1985, Rogerson 1991), or on few quantitative data on their occurrence in natural suspended detrital aggregates (Caron et al. 1982,1986, waters. They have been documented from a diverse Laybourn-Parry et al. 1991, Rogerson & Laybourn- array of freshwater habitats, and their ecology in those Parry 1992, Arndt 1993). These surfaces presumably ecosystems has been reviewed recently (Cook et al. provide both substrata for amoeboid locomotion as 1974, O'Dell 1979, Kyle & Noblet 1986, 1987, Lay- well as foci for microaggregations of attached bacteria bourn-Parry et al. 1991, Arndt 1993). Abundances of and other prey organisms. abundances up to free-living non-testate amoebae reported from fresh- several thousand-fold greater than in an equivalent water planktonic communities have ranged from volume of water have been demonstrated on sus- ~0.1ml-' up to several hundred ml-' based on the use pended detrital aggregates and at the aidwater inter- of Most Probable Number (MPN) cultural methods to face. Thus, the distribution of particulate material in estimate abundance. On average, these numbers are the plankton appears to be a major determinant in the modest reltiiive to the deiisities of he:ero:isphic nam- cccurrence ar?:! distribution of free-!i\ling arno~ha~in flagellates in fresh water (Berninger et al. 1991), but the plankton. most nanoflagellate abundances typically have been In this manuscript we report the occurrence of occa- obtained using direct counting methods and epifluo- sionally high abundances of naked amoebae in plank- rescence microscopy (Sherr et al. 1993). Interestingly, ton samples collected in the Black Sea during 1992 and cultural estimates of the abundances of heterotrophic 1993. Most samples had undetectable or low abun- nanoflagellates are typically 1 to 2 orders of magnitude dances of amoebae (<0.5 ml-l), but several samples less than the abundances of heterotrophic nanoflagel- contained abundances up to several hundred ml-l. lates obtained using direct counting methods, but These results confirm previous reports of amoebae in similar to the abundances of amoebae determined by this ecosystem (Kopylov & Sazhin 1988, 1989), and MPN culture methods (Caron et al. 1989). Based on indicate a close association of amoebae with parti- this type of reasoning, Arndt (1993) has concluded that culate material. Amoebae constituted a potentially the importance of naked amoebae, particularly small important, although highly patchy, component of species, has been underestimated in lake ecosystems the heterotrophic nanoplankton assemblages of this up to the present time. environment. The contribution of non-testate amoebae to marine plankton generally has been overshadowed by the importance of the more conspicuous sarcodine taxa METHODS within the , Foraminiferida, Polycystinea and (Caron & Swanberg 1990). Neverthe- Samples were collected during 5 cruises at stations less, amoebae have been observed from a wide range in the northern and eastern portions of the Black Sea in of marine environments, including estuaries, coastal May, July and September l992 and February and and open ocean pelagic ecosystems, and shallow and October 1993 (Fig. 1A). Up to 11 depths were exam- deep ocean sediments (Bovee 1956, Lighthart 1969, ined at each station. All water samples were collected Sawyer 1971, 1975a, b, 1980, Davis 1976, Sieburth et with 30 1 Niskin bottles. Hydrographic information al. 1976, Anderson 1977, 1994, Sawyer et al. 1977, (salinity, temperature, oxygen and pH) was obtained 1993, Davis et al. 1978, Burnett 1981, Caron et al. 1982, from the surface to the bottom at all stations. Samples 1986, Fernandez et al. 1989, Rogerson 1991, Alongi for ash free dry weight were collected on ~artorius~ 1992, Rogerson ?Y Laybourn-Parry 1992, Vsrs 1993a, b, filters (0.3 pm pore size). Filters were weighed prior to Butler & Rogerson 1995). Abundances of culturable filtration of the samples. Samples were then collected species of amoebae in marine plankton communities on the filters, rinsed and combusted at 500°C for 1 h have ranged from undetectable to nearly 200 ml-', but prior to reweighing the filters. abundances of 1-10 ml-' in estuarine/coastal waters to Microbial abundances and biomasses were deter- 1-10 1-' in oceanic waters have been more typical mined from light microscopical observations. Phyto- (Davis 1976, Davis et al. 1978, Rogerson & Laybourn- plankton abundance and taxonomic composition Parry 1992). (except phototrophic nanoplankton; see below) were Free-living amoebae from both freshwater and determined by light microscopy using an ~m~lival@ marine planktonic environments generally are as- microscope. Phytoplankton biomass (wet weight) was sumed to be associated with surfaces. Highest abun- calculated from abundances and biovolume estimates dances in the water column have been observed at the determined from the measurement of phytoplankton Murzov & Caron. Naked amoebae abundances 163

Fig. 1. Locations of (A) stations sampled for amoebae in the Black Sea during May 1992 and October 1993. and (B)stations at wh~chamoebae were observed during the 2 cruises cells and assuming specific geometric shapes clearly visible in some of the preparations for epifluo- (Makarova & Pishkilyi 1970, Senichkina 1986a, b). Bio- rescence microscopy and, where present, they were volunle of phytoplankton was converted to biomass enumerated along with the nanoflagellates. assuming a specific gravity of 1.0. Phototrophic and We attempted to estimate the efficacy of our direct heterotrophic nanoplankton abundances (2 to 20 pm counting method for determining the abundance of and protozoa, respectively) were determined naked amoebae by performing a laboratory experi- from glutaraldehyde-preserved (1% final concen- ment comparing direct microscopical counts and cul- tration) samples by epifluorescence microscopy using tural (MPN) counts for a cultured amoeba (Flabellula the primulin technique (Caron 1983). Biomass (wet hoguae; American Type Culture Collection #30733). weight) of the nanoplanktonic assemblages was deter- Amoebae were grown in liquid medium (natural, auto- mined from abundance estimates and volume esti- claved seawater) on bacteria enriched with 0.01% mates based on the measurement of cell dimensions, yeast extract broth and a sterile rice grain. Samples and assuming a specific gravity of 1.0. Estimates of the were collected from the culture during exponential biovolume of amoebae were exceedingly difficult be- growth of the amoeba and processed in parallel for cause of the irregular shape and variable thickness of epifluorescence microscopical counts using the tech- the cells. Estimations of amoeba biovolumes were nique employed for nanoplankton samples in the field obtained using measurements of the outline of the cells study and by the MPN technique Samples for MPN and thickness determined using a calibrated scale on cultural counts were diluted in a 2-fold serial dilution the focusing knob of the microscope. Bacterial abun- series using filter-sterilized culture water. Four repli- dances were obtained for some samples using the cates of each dilution were cultured in 96-well tissue acridine orange method (Hobbie et al. 1977). culture dishes uslng yeast extract broth to enrich the Amoeboid cells (irregularly shaped eukaryotic cells bacterial assemblage. Replicate plates were analyzed with clear unbroken margins, radiating or eruptive and the results combined to obtain an average MPN of , and clearly distinguishable nuclei) were amoebae in the original samples and to estimate the observed in preserved samples prepared and exam- error. Direct counts by epifluorescence microscopy of ined for nanoplanktonic using epifluorescence E hoguae in this analysis averaged 128 % of the MPN microscopy. Cells that did not display these features counts, but no significant differences were observed were not counted as amoebae. Identification of nano- owing to the large variability associated with the MPN planktonic cells as non-testate amoebae was con- estimates (average coefficient of variation = 38%). firmed by light microscopy on natural, live material Based on this analysis, we concluded that, for easily and on enriched samples to insure that the cells being recognizable forms, the direct counting method pro- enumerated by this method were amoebae. vided estimates of amoeba abundances that were com- Direct epifluorescence microscopy of amoebae is not parable to the MPN method. normally employed for enumerating these protozoa in The efficacy of the direct counting method applied to natural samples because of the difficulties of distin- field samples, however, is difficult to assess. The esti- guishing the cells from non-living material. Cultural mated limit of detection for the direct counting method estimates (MPN) are commonly used but were not used here was approximately 0.5 amoebae ml-' based possible in this study. Nevertheless, amoebae were on the amount of water filtered (up to 12 ml) and the 164 Aquat Microb Ecol 11: 161-169, 1996

portion of the filter examined (-30% of the filter area phytoplankton, and were presumably feeding on bac- covered by sample). Therefore, amoebae may have teria in the water and in the cultures. Biovolumes esti- been present in the samples at abundances <0.5 ml-' mated for this amoeboid form ranged up to approxi- but were undetectable by this method. This estimate mately 300 pm3. This morphotype was observed at also assumed that all amoebae present in the samples both shallow water and deep water stations in May could be identified as amoebae. We speculate that the 1992 and October 1993, with highest abundances direct counting method may have severely underesti- (9.6 rill-l) observed near the center of the eastern mated the abundances of amoebae c10 pm in size divergence at a depth of 25 m. At this depth and time, which can be important in natural samples (Butler & these amoebae constituted 8% of the total abundance Rogerson 1995). Some of these individuals probably of HNAN. Biomass calculated from these measure- were not distinguishable from detrital material be- ments indicated that amoebae constituted approxi- cause of the non-specific staining of some detrital par- mately 16% of the total biomass of HNAN in the sam- ticles with which the amoebae were often associated. ple with maximal abundance of amoebae. Amoebae Also, some amoebae retract pseudopodia and become constituted a greater percentage of HNAN biomass somewhat rounded when preserved. This behavior than of HNAN abundance because of the larger aver- cuuid make ihcm difficiili to lileii;i+ as amoebae ;ge biovo!umc of amoebae re!ative to other Eancr- in epifluorescence preparations. Therefore, the abun- planktonic protists. This calculation of the contribution dances reported here must be considered lower limit of amoeba biomass to HNAN biomass is based on our estimates of the actual abundances and biomasses of assumption of a single biovolume:biomass conversion non-testate amoebae in the samples. It was not possi- factor for all protists. This assumption is questionable ble to conduct comparisons between the direct count- given the differential effects of preservation on cell ing and cultural methods during the field study. shrinkage and the inherently different carbon contents of some protists (Verity et al. 1992, Stoecker et al. 1994). Therefore, this value can be considered only a RESULTS first-order approximation of the contribution of amoe- bae to HNAN biomass. The occurrence of naked amoebae during this study The second amoeba morphotype was larger (12 to was highly sporadic. Estimates of abundances of 20 pm) with short pseudopodia. Biovolumes estima- amoebae ranged from undetectable up to 380 ml-'. ted for these amoebae ranged up to approximately Amoebae were present in samples collected at 5 sta- 900 pm3. This morphotype was observed only during tions during May 1992 and at 9 stations during Octo- May 1992, and was observed at abundances of

(m)Amoeba Abundance (no.mlfl) (m) Temperature('C) (B) AmoebaAbundance (no.ml-l) (m)Temperature ("C) 0 2.5 5 7.5 10 0 5 10 15 0 - B ...... A 10- l020 i... E 20 -

30- n /,

30 30 40- i 50 - 40- 4040' 0 200 400 600 800 17.5 18 18.5 19 0 1000 2000 3000 400 1000 2000 3000 4000 5000

(*) HNANAbundance (no. rnl-l) (*)Salinity (%a) (*) HNANAbunbance (no. ml-1) (*) BacteriaAbundance (x106 ml-l)

(m) HNANBiomass (mg m-3) (m)Ash Free Dry Weight(mg I-') (W) HNANBiomass (mg m-3) (m)Ash Free Dry Weight(mg 1-l)

I 50-I I 0 5000 10000 15000 5 6 7 8 0 5000 l0000 15000 5 6 7 8 (*) PHYTBioniass (mg (*) Dissolved Oxygen(mg l-l) (4) PHYT Biornass (rng (*) Dissolved Oxygen(mg 1-l)

Fig. 2. Vertical profiles of (A) amoeba abundances and total Fig. 3. Vertical profiles of (A) amoebae abundances and total heterotrophic nanoplankton (HNAN) abundances, (B) tem- HNAN abundances; (B) temperature and bacterial abun- perature and salinity; (C) total biomass of HNAN and phyto- dances; (C) total biomass of HNAN and phytoplankton plankton (PHYT);and (D) ash free dry weight and dissolved (PHYT); and (D) ash free dry weight and dissolved oxygen oxygen concentration, conducted on 11 May 1992 at a station concentration, conducted on 16 May 1992 at a station at at 42" 30' N, 39' 00' E (water column depth = 2010 m). 'Abun- 44" 57' N, 36' 24' E (water column depth = 42 m) dance of HNAN in this sample was probably underestimated due to poor quality of filter preparations Positive, albeit weak correlations of abundances of amoebae were observed with HNAN abundances (Fig. 3D). Fragments of algae, whole algal cells and (Fig. 4C). A correlation coefficient of 0.60 was obtained amorphous detrital material were observed within the for the regression of log(HNAN abundance) versus vacuoles of these amoebae. The abundances of this log(amoebae abundance) with 1 anomalously high amoeba morphotype decreased with distance from data point excluded. Exclusion of the amoeba abun- Kerch Strait, and this form was not observed in waters dances from the HNAN data set did not greatly affect from the northwestern shelf area of the Black Sea. this relationship (correlation coefficient = 0.66). Simi- Correlations of abundances of amoebae with chemi- larly, amoebae abundances correlated weakly with ash cal, physical and biological parameters measured dur- free dry weight for a somewhat smaller subset of the ing the study were examined for those samples that data (correlation coefficient = 0.64 with 2 anomalously contained amoebae. No significant correlations were high values excluded). Ash free dry weight and bacte- obtained between the abundance of amoebae and rial abundance were not available for all stations and temperature, oxygen, salinity, phytoplankton abun- depths. dance or phytoplankton biomass. However, the high- est abundances of amoebae were observed in those samples that contained the highest abundances of DISCUSSION phytoplankton. Amoeba numbers also were not clearly correlated with the sampling depth (Fig. 4A) or with Previous studies of the HNAN of the Black Sea have the depth of the water column at the sampling sites emphasized the dominance of this assemblage by (Fig. 4B). Highest amoeba abundances, however, were zooflagellate species (Kopylov & Moiseyev 1983, Kopy- observed at the stations with the shallowest water lov & Sazhin 1989). However, naked, free-living amoe- columns. bae have occasionally been observed in the plankton of 166 Aquat Microb Ecol 11: 161-169, 1996

oxycline relative to abundances above or below that feature. Zubkov et al. (1992) has noted an abundant and unique protozoan fauna at the oxic-anoxic inter- face in the Black Sea, but the occurrence of naked amoebae apparently was not examined. The samples in this study were obtained within the oxygenated sur- face waters of the Black Sea, and the exact relationship between the oxycline and abundances of amoebae was not examined. High abundances of amoebae near the Sampling Depth (m) Depth of Water Column (m) base of the oxygenated layer at some stations in thls study may be indicative of elevated abundances asso- ciated with the oxycline (Fig. 3A), but no clear correla- tion was observed in regressions between abundances of amoebae and oxygen concentrations for the whole data set. 'vL'edic ~v~leidiiv~lswele ubse~vetiill iilis siudy vlliy with HNAN abundances and ash free dry weight. The correlation with HNAN (composed largely of hetero- trophic flagellates in most samples) was expected because of the common trophic activities of many of HNAN Abundance (no,d-l) PHYT Abundance (no. ml-l) the and amoeba taxa. Most nanoflagellates feed phagotrophically on bacteria and small algae. Fig. 4. Relationships between the abundance of amoebae and several biological and physical parameters for those samples Although many nanoflagellates feed on suspended in which amoebae were observed during cruises conducted in , a number of species graze micro- May 1992 and October 1993. (A)Depth at which each sample organisms attached to surfaces (Caron 1987). Increases was collected; (B) depth of the water column at the sampling in the density of bacteria, and attached bacteria in par- site (note: this does not specify the sampling depth); (C) total HNAN abundance including amoebae; (D)ash free dry weight ticular, would be expected to result in elevated abun- dances of both surface-associated heterotrophic nano- flagellates and amoebae. In addition, the amoebae the Black Sea. Kopylov & Sazhin (1989) noted that observed in this study were in the nanoplankton size amoebae occasionally constituted an important compo- class and thus contributed directly to HNAN abun- nent of the biornass of HNAN. Amoebae in that study dances, albeit modestly (maximum of 16% of total constituted up to 80% of the biomass of HNAN during HNAN abundance, usually < 10 %). May and June. No species descriptions were presented. We also found a weak correlation between the abun- Amoeboid cells have also been observed in coastal dances of amoebae and ash free dry weights of waters off Bulgaria (Kopylov et al. 1991) and in the sed- suspended particulate material in this study. Micro- iments of this area (Valkanov 1970). These obser- scopical examination of the fluorochrome-stained sam- vations, together with the results presented here, indi- ples showed that most amoebae were attached to par- cate that amoebae are probably a common component ticles in the plankton (primarily detrital aggregates). of the plankton of this inland sea, and that they can Therefore, a correlation with ash free dry weight was occasionally dominate the heterotrophic species within not surprj.sing. Previous studies have noted the associ- the nanoplanktonic size class. Dominance of other ation of naked amoebae with particulate material or marine nanoplanktonic protozoan assemblages by other surfaces (Davis et al. 1978, Caron et al. 1986, naked amoebae has not been reported, although such Laybourn-Parry et al. 1991, Rogerson & Laybourn- situations apparently are not uncommon in the benthos Parry 1992, Arndt 1993), and mobility and feeding by (Mare 1942, Butler & Rogerson 1995). most amoebae appear to be dependent on the avail- Correlations of abundances of amoebae with physi- ability of surfaces. cal, chemical or biological parameters in the plankton The weak nature of the correlation between the have been extremely rare because there are few quan- abundances of amoebae and ash free dry weight ob- titative data on the occurrence of these protozoa. served in this study is probably due to varying chemi- Abundances of amoebae in this study typically showed cal composition and age of the particulate material subsurface maxima (Figs. 2 & 3), although the surface sampled. The labllity of the organic compounds consti- microlayer was not examined. Kyle & Noblet (1986) tuting the particulate material should strongly affect noted that vertical distributions of amoebae in a fresh- the composition and extent of the microbial community water lake were somewhat higher in the region of the accumulating and growing on suspended detrital Murzov& Caron: Naked amoebae abundances

aggregates. In addition, a succession of microbial The use of a direct counting method for estimating species has been noted on suspended fecal material amoeba abundances, however, also has significant and detrital aggregates produced by marine zooplank- drawbacks. The numbers of amoebae reported here ton (Pomeroy et al. 1984, Caron et al. 1986, Davoll & may underestimate the actual abundances of amoebae Silver 1986). Therefore, the extent of development of because of the difficulties of distinguishing amoebae in the microbial assemblage present on suspended detri- preserved samples. This possibility is particularly high tal material at any particular time should be a conse- for very small amoebae (

Acknowledgements. The authors are grateful to the crews of Caron DA, Swanberg NR (1990) The ecology of planktonic the research vessels of the Ukranian Scientific Center of the sarcodines. Rev Aquat Sci 3:147-180 Ecology of the Sea, and to Prof Dr E Z. Samishev, Dr N. V Cook WL, Ahearn DG, Reinhardt DJ, Reiber RJ (1974) Blooms Kovalyova. Dr S. A. Scrgin and graduate student Ju. V of an alrjophorous amoeba associated wlth Anabaena in a Bryantseva for data concerning ash free dry weights, bacter- fresh water lake. Wat Air Soil Pollut 3:71-80 ial and phytoplankton abundance and biomass. Ee Lin Lirn Daggett PM, Sawyer TK, Nerad TA (1982) Distribution and provided technical assistance with the laboratory study com- possible interrelationship of pathogen~cand nonpatho- paring the direct counting and MPN methods. We also thank genic Acanthamoeba from aquatic environments. Microb Elena A. Kondratyeva for translation. 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Responsible Subject Editor: T. Fenchel, Helsingor, Denmark Manuscript first received: August 2, 1995 Revised version accepted: June 13, 1996