J. Euk Mrc,ohiol., 46(2), 1999 pp. 160-164 0 1999 by the Society of Protozoologists

Abundance of Naked Amoebae in Sediments of Hiroshima Bay, Set0 Inland Sea of Japan

OLIVIER DECAMP,' MUTSUMI TSUJINO and TAKASHI KAMIYAMA Coustul Environment and Productivity Division, National Research Institute of Fisheries and Environment of Inland Sea, Ohno, Saeki,

Hiroshima 739-0452, Japan

ABSTRACT. The present paper provides the first data on naked amoebae from sediments of Hiroshima Bay. Three stations in the inner part of the bay were sampled over a three-month period. Abundance of naked amoebae ranged from 1,019 to 45,561 cells/g dry sediment. Results indicate: (i) surface sediment populations in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May; and (iii) the site located near Hiroshima City had fewer amoebae on several occasions than the other two sites. There was a negative exponential relationship between acid-volatile sulfide concentration and abundance of amoebae. Most amoebae were small with the average size ranging from 6.6-14 pm. Morphotype 1, amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, were dominant (40-1 00% of enumerated amoebae). Morphotypes 2 and 3 (limax amoebae) were found in lower numbers than the other two morphotypes. The proportion of amoebae occupied by Morphotype 4 (fan-shaped or discoidal-flattened amoebae) was higher at a lower total abundance.

Supplementary key words. Distribution, ecology, estuaries, morphology, protists, protozoa, sediment microbial communities.

AKED amoebae have been reported from a range of ma- N rine locations including estuarine and oceanic waters, benthic and shore sediments, and surface of macroalgae [2-6, 8, 22, 251. However, it is only recently, with the documentation of high numbers of amoebae, that studies have considered their ecological role. For example, up to 3.2 X lo6 amoebae/m2 have been reported from seaweed surfaces [22] and up to 43,000 cells/liter have been reported from estuarine waters [23]. These impressive numbers suggest that amoebae may play an impor- tant role in carbon flow in estuaries.

Naked amoebae have also been reported to be abundant in a variety of marine benthic habitats such as estuarine sediments [4] or deep-sea sediments [6]. The ecology of naked amoebae has recently been investigated in marine sediments of the Clyde Sea [7] and Bermuda [2] using a method providing information on abundance and diversity 131. High abundances were record- ed: up to 14,883 amoebae/cm3 in the Clyde Sea [7] and 28,761 amoebae/g dry sediment in Bermuda marine sites [2].

Temperature is thought to be the major environmental param- eter controlling temporal variation in abundance and diversity of amoebae in the water column [ l , 31. In sediment, however, consistent temporal variation in abundance has not been ob- served [7, 111. It has been suggested that interactions in sedi- ment might be more complex than those in the water column. Therefore, one of the aims of this paper was to gain a first impression of numbers of naked amoebae at three different sites in the sediment of this bay. Considering the numerical impor- tance of small-sized amoebae in algal stands, water column, and sediments 13, 221, particular attention was paid to these amoe- bae.

To our knowledge, there are no reports on free-living amoe- bae inhabiting Japanese marine sediments. The present paper provides the first data on naked amoebae from sediments of the Seto Inland Sea of Japan.

MATERIALS AND METHODS Study sites. Three sampling stations in the inner part of Hi-

roshima Bay, Set0 Inland Sea of Japan, were studied (Fig. 1). Station 1 was near the coast (34" 20.49 N, 132" 22.82 E) at a depth of 13.5 m. Station 2 was near the island of Ninoshima (34" 18.40 N, 132" 24.22 E) at a depth of 21.2 m. Sediment in these two stations was silty-clay. Station 3 was located towards the outer bay (34" 15.92 N, 132" 24.80 E) at a depth of 24.5 m where the sediment was clay-sand.

I To whom correspondence should be addressed. Telephone: 81 -829- 55-0666; FAX: 8 1-829-54-1216; Email: decamp@nnf.affrc.go.jp and olivier.decamp@ mailexcite.com

Sample collection and analysis. Water and sediment sam- ples were collected in March, April, and May of 1998. Water salinity, temperature, and dissolved oxygen concentration pro- files were measured on the SET0 RV with a portable Salinity- Temperature meter (YEO-CAL Model 602-MKII, Australia) and DO meter (TOA Electronics Ltd. model DO-25A, Kobe, Japan). Water samples were collected at a depth of 1 m above the sediment using a Niskin bottle. A subsample (20 ml) was immediately fixed with 2% formalin and brought back to the laboratory for the enumeration of bacteria. The abundance of bacteria was estimated by epifluorescence following the proto- col described by Hobbie et al. [12]. Bacteria on two filters were counted per sample.

Sediment was sampled with a 4-cm diam. KK-type gravi- metric core sampler (Hashimoto Scientific Co., Kyoto, Japan) [15]. At each station, two cores were taken for physico-chem- ical analysis. Cores were divided into two layers: surface (0-1 cm) and subsurface layer (1-2 cm). Each sample was analyzed for water content, ignition loss, and acid-volatile sulfide. Igni- tion loss was estimated as weight loss on ignition at 550" C for 6 h [24]. Acid-volatile sulfide concentrations were measured with Hydrotech-S gas detection tubes (Gas Tec, Inc., Newark, California, USA).

One core was used for the enumeration of free-living amoe- bae. Sediments from the surface and subsurface layers were diluted in bottles containing 750 ml of autoclaved seawater from Hiroshima Bay. Bottles were then brought back to the laboratory for enumeration. Free-living amoebae were enumer- ated following a method described by Anderson and Rogerson [3] and Butler and Rogerson [7]. Briefly, aliquots of 5 +1 and 10 p1 of diluted sediment samples were deposited in 2 ml of modified Erdschreiber Medium (Merds) [ 171, contained in each well of a 24-well microplate (Sumilon, Sumitomo Bakelite Co., Ltd, Tokyo, Japan). Two microplates were used per assay, with a total of 48 wells. Plates were incubated in the dark at 18" C , then checked after 2-3 wks for the presence of free-living amoebae. Ideally, these populations should develop from a sin- gle cell. Samples were therefore diluted in such a way that amoebae were not present in all inoculated wells. Amoebae were classified into four morphotypes during determinations of abundance and diversity using Anderson and Rogerson 131 as follows: Type 1-amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, including genera such as Acanthamoeba, Mayore l la and Vexil l i fera; Type 2- limax amoebae with non-eruptive locomotion, including genera such as Hartmannel la and Saccamoeba; Type 3-Iimax amoe- bae with eruptive locomotion, including genera such as Vahlk- ampjia, and Heteramoeha; and Type 44 i sco ida l or fan-shaped

160

DECAMP ET AL.-NAKED AMOEBAE IN THE SET0 INLAND SEA 161

March

1

I 2

3 - 0 2 4 6

April May

0 2 4 6 0 2 4 6

Abundance of amoebae (104 cells/g dry sediment)

Fig. 1 . Location of sampling in Hiroshima Bay and abundance of naked amoebae in surface (0) and subsurface (0) layers of each station in March, April, and May 1998 (Bar = standard deviation). Some standard deviations are too small to be visible on the graphs.

flattened amoebae, including genera such as Platyamoeba and Vannella.

Free-living amoebae were filmed with a Sony CCD-IRIS camera attached to an Olympus IX-70 inverted microscope and taped with a Ikegami TVR-7480 Time Lapse Video Cassette Recorder. The size of naked amoebae was estimated from re- corded specimens.

RESULTS Environmental data. Salinity, dissolved oxygen, and tem-

perature of the water layer 1 m above sediment showed no variation between sites. There were temporal variations: an in- crease in water temperature from March to May of 3.7-4.2" C and a decrease in dissolved oxygen and salinity over the same period (Table 1). The average abundance of bacterioplankton ranged from 1.19-2.28 X lo6 cells/ml, and showed no clear geographical trend. The abundance of bacterioplankton in Sta-

Table 1. Physicochemical characteristics and abundances of bacteria in the water layer 1 in above the bottom of sediments at three stations in Hiroshima Bay (see Fig. I ) .

Stations

Month 1 2 3

Temperature ("C) March 11.4 11.3 11.2 April 12.6 12.6 10.9 May 15.1 15.2 15.4

Salinity ( g o ) March 32.8 32.9 32.9 April 32.5 32.1 32.8 May 32.3 32.5 32.4

DO (mgfl) March 6.8 6.5 7.1 April 5.1 5.3 5.3 May 4.1 5.3 5.4

Bacteria (106/ml) March 1.19 1.42 1.38 April 2.28 1.7 I I .48 M aY 1.63 1.98 1.64

tions 2 and 3 seemed to increase slightly from March (Table

Variations among sites were found regarding the sediment characteristics (Table 2) . Higher ignition loss was consistently recorded in Station 1 while lower ignition loss was recorded in Station 3. The highest concentration of sulfide was recorded in Station 1 in March. These data indicate a eutrophication gra- dient, with Station 1 more enriched in organic matter than Sta- tions 2 and 3.

Numbers of amoebae. Abundance of naked amoebae fluc- tuated greatly, ranging from 1,019 (Station 1, subsurface) to 15,517 (Station 2, surface) cells/g dry sediment in March; from 7,530 (Station 1 , subsurface) to 39,256 (Station 3, surface) cells per/g dry sediment in April; and from 4,139 (Station 3, sub- surface) to 45,561 (Station 2, surface) cells/g dry sediment in May. These results indicate that (i) surface sediment popula- tions in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May; and (iii) the coastal site, Station 1, had fewer amoebae on several occasions than the other two sites (Fig. 1). There was a negative exponential relationship between sulfide and abundance of amoebae, with the lowest abundance of amoebae recorded at the highest sulfide concentration of 1.61 mg/g dry sediment in subsurface station in March (Fig. 2). At lower sulfide concentrations (< 1 mg/g dry sediment), higher abundances of amoebae were generally observed at higher water temperature. There was a linear rela- tionship (r = 0.761) between bacterial abundance (log cells/ml seawater) in the water layer above sediment and amoeba abun- dance (log cells/g dry sediment) in the surface layer.

Morphotypes of amoebae. Amoebae communities were dominated by Type 1 amoebae, with their proportion ranging from 47-100% of total amoebae. Type 4 amoebae were the second most common group in all samples in March, and in several samples in April and May. Limax amoebae, Types 2 and 3, were almost absent from sediment in March. There was

1).

162 J. EUK. MICROBIOL., VOL. 46, NO. 2, MARCH-APRIL 1999

Table 2. Sediment characteristics of surface (0-1 cm) and subsurface (1-2 cm) layers. The percentage size fraction of sediment <63 pm was estimated by sieving in March (first number) and April (second number).

Stations

1 2 3

Month 0-1 cm 1-2 cm 0-1 cm 1-2 cm 0-1 cm 1-2 cm

Sediment <63 p,m (%) Water content (%) March

April May

May

May

Ignition loss (%) March April

Acid volatile sulfide (mg/g) March April

silty clay 8 1-95 80 78 79 13.5 15 14

1.155 0.192 0.473

silty clay 81-95 78 72 74 13.2 14.3 13.7 1.608 0.549 0.741

silty clay 91-95 76 77 72 11.6 14.3 13 0.29 I 0.16 0.301

silty clay 9 1-95 73 73 71 11.9 13.2 12.4 0.45 0.28 0.292

clayey sand 80-80 75 69 65 I 1 1 1 11.8 0.154 0.314 0.205

clayey sand 80-80 65 64 62 9.7

12.3 10 0.315 0.37 1 0.295

a clear, linear relationship between total abundance of naked amoebae and abundance of Type 1 (r = 0.972), whereas the relationship between total abundance of naked amoebae and abundance of Type 4 was weaker (r = 0.690). The slopes of the linear regression between total abundances and type abun- dances were steeper for Type 1 than for Type 4 (0.990 vs. 0.413). As a result, the ratio in abundance of Type 1 to abun- dance of Type 4 increased with total abundance of amoebae. Ratios were 0.89 and 1.7 at abundance of 2.2 and 7.5 X LO3 cellsig dry sediment respectively, and 10.5 and 16 at abundance of 1.5 and 4.6 X 104 cells/g dry sediment, respectively. The proportion of the community occupied by Type 4 amoebae was therefore higher at lower total abundance (26.7% at 4.14 X lo3 cells/g dry sediment) than at higher total abundance (5.1% at 5.6 X 104 cells/g dry sediment).

In the surface layer, Type 1 amoebae were, on average, 6.5X more abundant than Type 4, whereas in the subsurface layer,

h c C

E" u)

'0 2

P - Q 0 m 0

Q m

0

m 0 Q 0 S m U C 3

- v

-8 E c

n a

5.0

4.5

4.0

3.5

3.0 c

0.0 0.5 1 .o 1.5 2.0

Acid volatile sulfide (mg/g dry sediment)

Fig. 2. Relationship between abundance of amoebae (log cells/g dry sediment) and acid-volatile sulfide (mg/g dry sediment). Equation of the linear regression: y = - 0.819X + 4.378 (r = 0.709). March sam- ples (U), April samples (O), May samples (A).

Type 1 were on average 4.3X more abundant than Type 4. There was a negative correlation (r = -0.651) between acid- volatile sulfide concentration and abundance of Type 1 amoe- bae. There was no significant correlation between sulfide and abundance of Type 4 amoebae. Most amoebae were relatively small with the average size ranging from 6.6 pm (Type 4) to 14 pm (Type 1).

DISCUSSION

The abundances of amoebae in Hiroshima Bay were similar or higher than abundances reported in the literature: 2,335- 6,850 cells/g dry fine sediment off Plymouth (Mare, 1942, re- ported in Butler and Rogerson [7]); 2,224 cells/cm3 fine sedi- ment and 874 cells/ cm3 in sandy sediment of the Clyde Sea [7]; 28,761 and 17,597 cells/g dry sediment in Mullet Bay, Bermuda [2]. Abundance has been reported to decrease with depth [7], possibly related to an increase in reducing conditions with depth or lower porosity.

Temperature influences total abundance of amoebae in the water column, with higher abundance recorded at higher tem- perature [I , 31. The lowest abundance of amoebae in Hiroshima Bay was indeed recorded in the coldest month (Fig. 1) . How- ever, temperature alone did not govern abundance in sediments of Hiroshima Bay, as in sediments of the Clyde Sea [7]. Benthic protists are thought to be influenced by a variety of factors, such as rapid growth of populations, immigration and emigra- tion of species, predation, effect of water current movements, bioturbation, depletion of resources, and variation in food avail- ability [18]. The present study provides information on possible relationships between the abundance of amoebae and some of these factors.

Hiroshima Bay is a eutrophic embayment of the Seto Inland Sea and it is strongly influenced by riverine input, of 4.1 m3/ (m2 . yr) [30]. As a consequence of that freshwater input, het- erogeneities occur within the bay: (i) concentrations of partic- ulate organic carbon in the surface layer are higher near the coast than towards the outer bay; (ii) concentrations of bacter- ioplankton in surface layers are higher near the coast (O.D., unpubl. data); and (iii) settling fluxes are governed by vertical circulation of water rather than gravity, and are therefore higher towards the outer bay than near the coast [26]. Bacterioplankton abundance was typical of eutrophic waters and within the range previously reported in Hiroshima Bay [9]. Higher ignition loss and acid-volatile sulfide concentration in sediment at Station 1 indicate stronger eutrophication in the coastal area, as also re- ported by Rajendran et al. [20, 211.

DECAMP ET AL.-NAKED AMOEBAE IN THE SET0 INLAND SEA 163

Bacterial abundance of 0.6-1.8 X lo7 cells/g [21] to 7-8 X lo7 cells/g [20] has been reported in inner Hiroshima Bay. Al- though eutrophication is stronger in the coastal area, bacterial abundance has been reported to be higher near our Station 2 than near our Station 1 (coastal area): 0.61 X los cells/g vs. 0.14 X los cells/g [19] and 1.5 X lo8 cells/g vs. 0.8 X lo8 cells/g [20]. That higher bacterial abundancehiomass would then support a higher abundancehomass of phagotrophs, i.e. protozoa and nematodes. Spatial variations in food availability, although not estimated in the present study, would be consistent with the higher abundance of amoebae (present study) and nem- atodes (data for March only; M.T., unpubl. data) in Stations 2 and 3 compared to Station 1.

Abundance of amoebae during this three-month period was negatively correlated with acid-volatile sulfide. The impact of sulfide on the composition and diversity of macrobenthic com- munities is well documented [29]. To our knowledge, this re- lationship has not been reported for benthic naked amoebae. Anaerobic naked amoebae have recently been described in ma- rine sediment [27]. Higher sulfide concentration has consis- tently been reported in coastal areas near Station 1 [13, 281. Anaerobic or microaerophilic amoebae might therefore have been present in Station 1, but would not have grown under the aerobic enrichment conditions used in the present study. An enumeration method focusing on anaerobic microorganisms, such as that described by Smirnov and Fenchel [27], would provide useful information.

Type 1 amoebae, with one exception, were more abundant than the other three morphotypes. However, at a lower abun- dance of amoebae, Type 4 became more important and were even more numerous than Type 1 (Station 1, surface in March). Morphotype 1 did tend to be larger than Morphotype 4 during this investigation (14 pm vs. 6.6 pm), as observed by Anderson [l]. The smaller size of Type 4 might explain why the abun- dance of Type 4 decreased less with depth than Type 1. Butler and Rogerson [7] reported that limax amoebae were abundant in Clyde sediment ( 2 35%) and explained this finding by their shape being suited to migration through small interstices. Dur- ing the present study, the proportion of limax forms (Types 2 and 3) was very low in March (0-8%), and increased in April and May. However, the proportion of limax amoebae showed strong fluctuations between sites and depth, being as low as 4% (Station 1, subsurface in May) and as high as 48% of total amoebae (Station 2, subsurface in May). The lower average abundance of limax forms observed in the present study (7.7% of counted amoebae) requires further investigation. Potential explanations for this difference include sediment characteristics (muddy in Hiroshima Bay vs. sandy in Clyde Sea), range of temperature, and nutrient characteristics (eutrophic bay of the Set0 Inland Sea vs. Scottish Sea Loch).

The actual population of amoebae is likely to have been un- derestimated by the present method. Major limitations of this enrichment method include the possibility that some amoebae cannot be cultured and the presence within a sample aliquot of more than one cell [3, 71. Despite its limitations, the present method gave higher counts than other enrichment methods [7]. Direct enumeration, although preferable, would have its limi- tations too; fixed amoebae, and especially small-size amoebae, which are known to be abundant in marine sediment [present study, 2, 71, would have been difficult to recognize and separate from fine detritus. Indeed, Luftenegger et al. [16] reported that the efficiency of the direct method to estimate number of mi- croscopic soil organisms decreased with decreasing body size. Another source of underestimation is due to the incubation con- ditions, i.e. aerobic and in the dark. The method is thus restrict- ed to aerobic or microaerophilic, bacterivorous amoebae. An-

aerobic amoebae, such as those reported from the upper 5 cm layer of sediment near Helsingor, Denmark [27], and algivorous amoebae would not be able to grow under the incubation con- ditions used in the present study.

The present study showed that temperature and sulfide were two important sediment parameters controlling total abundance and morphotype of amoebae. Similar variation in bacterial abundance between sites (i.e. bacterioplankton in the water lay- er above sediment in the present study), in sediment bacterial biomass [20, 211, in abundance of amoebae (present study), and in the abundance of nematodes (M.T., unpubl. data) would con- firm the complexity of interactions between the abundance of benthic naked amoebae and environmental parameters. Biotur- bation due to nematodes creates oxic microhabitats, which are available to protozoons [ 101. Moreover, nematodes have been shown to influence microbial growth [14], and thus increase the food availability. Further work is required in order to under- stand the relationship between abundance of amoebae and mor- photype diversity and factors such as food availability and com- petition with other protists and nematodes.

ACKNOWLEDGMENTS This work was supported in part by a grant from the Science

and Technology Agency of Japan. We thank Dr. K. Tamai and Dr. S. Arima for their precious help with this manuscript and Mr. Goto, Captain of the RV Seto.

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Received 8-24-98, 11-26-98, 1-14-99: uccepted 1-17-99

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