Trophic coupling of a testate amoeba and Microcystis species in a hypertrophic pond

Limnology (2004) 5:71–76

? The Japanese Society of Limnology 2004

DOI 10.1007/s10201-004-0114-9

Yuichiro Nishibe · Pathmalal M. Manage Zen’ichiro Kawabata · Shin-ichi Nakano

Received: September 10, 2003 / Accepted: January 18, 2004

Y. Nishibe 1 (*) · P.M. Manage

Center for Marine Environmental Studies, Ehime University,Matsuyama, Japan

Z. Kawabata

Center for Ecological Research, Kyoto University, Otsu, Japan S. Nakano

Faculty of Agriculture, Ehime University, Matsuyama, Japan Present address:1

Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1Minato-cho, Hakodate 041-8611, Japan Tel. ?81-138-40-5543; Fax ?81-138-40-5542e-mail: nishibe@?sh.hokudai.ac.jp

Abstract Seasonal changes in abundance of the testate amoeba Penardochlamys sp. and its food vacuole contents were investigated in relation to blooms of the cyanobacteria Microcystis spp. in a hypertrophic pond from April 1999 to March 2000. The behavior of the amoeba feeding on M .aeruginosa and M . wesenbergii was also observed in the laboratory. The amoeba was detectable from late May to November 1999 during the blooms of Microcystis spp. Cell densities of the amoeba ?uctuated between 1.4 and 350 cells ml ?1 with some sporadic peaks, which did not coincide with rapid decreases in the abundance of Microcystis spp. Food vacuoles contained only Microcystis cells; other prey items were not found, suggesting that this amoeba utilized only the cyanobacteria as food. The amoeba was frequently found attached to Microcystis colonies, but was not associ-ated with other suspended particles. Observation of the amoeba feeding revealed the feeding mechanism and that the amoeba was able to graze on both species of Microcystis . These results suggest that the trophic coupling of these organisms is substantial, although grazing by the amoeba is not suf?cient to regulate the dynamics of Microcystis populations in this hypertrophic pond.Key words Testate amoeba · Microcystis · Grazing · Food vacuole contents · Trophic linkage

Introduction

Protistan grazing is now recognized to be an important process in the loss of cyanobacterial abundance in fresh-water and marine systems (Dryden and Wright 1987; Caron

et al. 1991; S

ˇimek et al. 1996, 1997). Rhizopods, consisting of naked and testate amoebae, common members of the protistan community in aquatic habitats (Caron and Swanberg 1990; Arndt 1993; Muylaert et al. 2000; Song 2000), have long been known as consumers of cyano-bacteria (reviewed by Dryden and Wright 1987). Previous laboratory studies have demonstrated that some rhizopod species are very active grazers with a strong tendency to select speci?c prey, particularly cyanobacteria (Ho and Alexander 1974; Wright et al. 1981; Huang and Wu 1982;Yamamoto and Suzuki 1984; Daft et al. 1985; Becares and Romo 1994). This evidence suggests that rhizopod grazing can have a signi?cant impact on the dynamics of natural cyanobacterial populations. In fact, Cook et al. (1974) re-ported that a mayorellid-like amoeba ingested exclusively Anabaena planktonica and suggested that their grazing was responsible for the termination of the cyanobacterial bloom in a eutrophic lake.

The cyanobacterium Microcystis is often the dominant component of blooms in eutrophic and hypertrophic lakes, ponds, and reservoirs. There are some reports of rhizopods grazing on Microcystis in laboratory experiments (Yamamoto 1981; Yamamoto and Suzuki 1984) and ?eld observation (Whitton 1973), but little is known about the seasonal abundance of any particular species of rhizopod that is capable of grazing on Microcystis . Furthermore, in-formation about the feeding habits of these rhizopods, an important aspect for understanding their trophic roles in natural environments, is quite limited.

In the fall of 1998, we found a testate amoeba,Penardochlamys sp. (Testasealobosia: Arcellinida), col-lected from a hypertrophic pond in Japan, to be a grazer of Microcystis by microscopic observation of the food vacuole contents (Fig. 1). Some morphological characteristics of Penardochlamys sp., such as an ovoid or round test with a

wavy border, closely resemble those of Penardochlamys arcelloides (Penard, 1904), but our Penardochlamys sp. has a single nucleus and its cell diameter ranges from 20 to 25μm (Fig. 1), whereas P . arcelloides is a binuclear species 60–70μm in diameter (Penard 1904; Bovee 1985). We there-fore identify the testate amoeba investigated here simply as Penardochlamys sp.

In the present study, we investigated the seasonal changes in abundance and food vacuole contents of the testate amoeba Penardochlamys sp. in a hypertrophic pond in Japan. The behavior of the amoeba feeding on Microcystis spp. was also observed in the laboratory, and trophic coupling between the amoeba and Microcystis blooms is discussed.

Materials and methods

Field investigation

Furuike Pond (33°49?N, 132°48?E) is a hypertrophic im-poundment located in Sancho, Matsuyama City, Ehime Prefecture, Japan. The pond has a surface area of ca.7400m 2 and a maximum depth of 1.5m. The physical,chemical, and biological characteristics of this pond have already been reported by Nakano et al. (1998, 2001) and Hirose et al. (2003).

Water samples were collected from the surface with a bucket at the near-shore station (see Fig. 1 in Nakano et al.1998) once a week from April to September 1999, January to March 2000, and twice a week from October to Novem-ber 1999. All samples were taken at around the same time

of day (0930–1030). Water temperature and pH were mea-sured simultaneously with a thermistor pH meter (TOA Electronics, Tokyo, Japan).

For enumeration of Penardochlamys sp., a 100-ml por-tion of the water sample was ?xed immediately with acidi-?ed Lugol’s solution at a ?nal concentration of 1% and concentrated by natural sedimentation. Cells of the testate amoeba were counted in a hemocytometer under a light microscope at a magni?cation of ?400. Another 100-ml portion of the water sample was ?xed and concentrated as described above for enumeration of phytoplankton. Each species of phytoplankton was identi?ed and counted in a hemocytometer under a light microscope at a magni?cation of ?400. Identi?cation of Microcystis species was based on cell size, colony form, and sheath characteristics. After the enumeration, each sample was slightly sonicated (20kHz,2–3min) to break up colonies of Microcystis species, and the cyanobacterial cells were counted as described above.

To examine the food vacuole contents of Penardo-chlamys sp., a 100-ml portion of the water sample was immediately ?xed with 20% glutaraldehyde at a ?nal concentration of 1% and then concentrated by natural sedi-mentation. A 0.05-ml aliquot of the concentrated sample was mounted on a glass slide, and 13–55 amoeba cells were observed under a light microscope at a magni?cation of ?400–600. All prey particles in the food vacuoles were identi?ed and counted. We also checked whether the amoeba cells inspected were attached to a substrate or were freely suspended.

Observation of feeding behavior

Penardochlamys cells attached to colonies of M . aeruginosa and M . wesenbergii were continuously observed using a microscope to acquire direct evidence of grazing on the two cyanobacteria and to elucidate the feeding mechanism.Water samples for this were collected on June 1, August 24, September 26, and October 21, 2000, by the same procedures as described for the ?eld investigation and were transferred to the laboratory within 0.5h of collection.Colonies of both cyanobacteria species carrying living Penardochlamys cells were then picked out from the water sample using capillary pipettes and transferred into a hemocytometer. This hemocytometer was placed under a light microscope equipped with a CCD camera (C4742-98,Hamamatsu Photonics, Hamamatsu, Japan) at a magni?ca-tion of ?400. Observations were carried out at room tem-perature (25°C), but changes in water temperature in the hemocytometer during the observation period were mini-mized by using a microwarm plate (MP-10DM, Kitazato Supply, Fuji, Japan). One single cell of Penardochlamys sp.was inspected in each observation. Simultaneous observa-tion and analysis of the recorded images of feeding behavior were done on the monitor using image analysis software

(Aquacosmos, Hamamatsu Photonics).

Fig. 1.A freely suspended cell of Penardochlamys sp. collected in Furuike Pond, with four ingested Microcystis cells. Glutaraldehyde ?xation, bar 5μm

73 Results

The surface water temperature increased with some ?uctua-

tions from April (14.4°C) to late August (28.8°C) and then

decreased until late December 1999 (4.8°C). From January

to February 2000, the temperature ranged from 6.2°C to

9.4°C and increased again in March. The pH varied between

7.8 and 10.7 from April to December, decreased rapidly in

January, and increased again from February onwards.

The two species of Microcystis(M. aeruginosa and

M. wesenbergii)predominated in the phytoplankton

community from May to November, and heavy blooms

were observed during this period. The cyanobacteria

Merismopedia spp. and the diatom Synedra berorinensis

were also abundant during the period. The cyanobacteria

Phormidium mucicola and Anabaena sp. dominated during

April and early May, respectively. From December

until early March, the dominant phytoplankters were

chlorophytes, such as Kirchneriella spp. and Scenedesmus

spp.

Several sharp peaks in the cell densities of M. aeruginosa

and M. wesenbergii were detected from May to October,

and the maximum density reached 12.7 ? 106 and 2.2 ? 106

cells ml?1 on May 26, respectively (Fig. 2A). M. aeruginosa

contributed on average to 90% of the total cell density of

Microcystis from April to August, but thereafter the per-

centage of M. wesenbergii was relatively high, accounting for 16%–65% of total Microcystis abundance.

The abundance of Penardochlamys sp. was measured from May 26 to November 30, 1999 (Fig. 2B). During this period, cell densities of the testate amoeba ?uctuated be-tween 1.4 and 350 cells ml?1, with several higher densities on May 26, July 14, September 1, October 16, and October 26 (Fig. 2B). We could not detect the presence of testate amoeba from April 1 to May 19, 1999, or from December 8, 1999, to March 29, 2000 (Fig. 2B). We examined the rela-tionship between abundance of Penardochlamys sp. and abiotic and biotic factors within the periods when the amoeba was present, but we found no signi?cant correlation between cell densities of the testate amoeba and water tem-perature, pH, cell density of M. aeruginosa, or the cell den-sity of M. wesenbergii.

In total we examined 1397 cells of Penardochlamys sp. and the food vacuole contents. Between 80% and 100% (average, 96%) of the Penardochlamys cells had food vacuoles containing phytoplankton cells, all of which were identi?ed as Microcystis spp., although we could not dis-criminate whether the ingested cyanobacterial cells were M. aeruginosa or M. wesenbergii. No other phytoplankton cells were observed, nor were any other prey items such as other protists or detritus found. Around the Penardochlamys cells there were many egested indigestible remains of Microcystis cells that had turned a deep magenta and had become smaller than living cells (see Fig. 3A), indicating that Penardochlamys sp. could digest part of the Microcystis cells. The average numbers of ingested Microcystis cells per amoeba on each sampling day varied from 3.2 to 9.1 cells (mean ? SD, 6.1 ? 1.2), tending to be higher from late June to early September but lower during October and November.

The testate amoeba was found attached only to colonies of M. aeruginosa (Fig. 3A), and M. wesenbergii (Fig. 3B), but not to other substrates.More amoebae were found attached to Microcystis colonies than were found freely suspended (Student’s t-test, p?0.05). Freely suspended amoeba cells were always found when testate amoeba cells were present. With regard to the attached cells of Penardochlamys sp., the percentages of amoeba cells at-tached to M. aeruginosa colonies were relatively high with ?uctuations between 81% and 100%, decreasing markedly on August 18 (50%) and further decreasing from that day until early September. After that, the ratio gradually became higher. In contrast, amoeba cells attached to M. wesenbergii were rare until mid-August, remained high from August 18 to November 5 (50%–100%), and gradually decreased from then onwards.

With microscopic observation, ten feeding events in 12 amoeba cells with M. aeruginosa and six events in 10 amoeba cells with M. wesenbergii, respectively, were identi-?ed. The feeding behavior of the amoeba proceeded as follows: (1) the amoeba intruded a ?nger-like pseudopo-dium into a colony of cyanobacterium, (2) the pseudopo-dium made contact with a single cyanobacterial cell using the tip of the pseudopodium and engulfed the cell, (3) the engulfed cyanobacterial cell was then transported below the pseudostome of the main body of the amoeba, and (4) the cyanobacterial cell was ingested and formed a food vacuole. No differences were observed in the feeding be-havior when the prey was M. aeruginosa or M. wesenbergii. Digestion and egestion processes were also observed: the Fig. 2.A Seasonal changes in cell densities of Microcystis aeruginosa (solid line) and M. wesenbergii (broken line) and B seasonal changes in cell densities of Penardochlamys sp. in Furuike Pond during the study

period

food vacuoles containing Microcystis cells moved randomly within the amoeba cell during digestion, and several indi-gestible remains of Microcystis were simultaneously egested from the pseudostome.

Discussion

Previous studies have reported that some species of naked amoeba could graze on Microcystis species. Whitton (1973)brie?y noted that a small, naked amoeba found in the muci-laginous mass of the cyanobacterium grazed on Microcystis spp. Also, Yamamoto (1981) and Yamamoto and Suzuki (1984) found that the naked amoeba Nuclearia sp. could feed on M . aeruginosa , M . elabens , and M . ?os-aquae in the laboratory. In the present study, from observations of feeding behavior, it appeared that the testate amoeba

Penardochlamys sp. was capable of grazing on both M .aeruginosa and M . wesenbergii . In addition, the results of investigating food vacuole contents indicated that the testate amoeba utilizes only Microcystis as food in Furuike Pond. As far as we are aware, this is the ?rst report which notes grazing on Microcystis by a testate amoeba.

The results in the present study, that food vacuoles of Penardochlamys sp. contained only Microcystis cells and that no amoeba cells attached to particles other than Microcystis colonies, suggested that the amoeba has a spe-ci?c preference for Microcystis species. Cook et al. (1974)and Becares and Romo (1994) also reported that some rhizopods collected from natural waters seemed to have strong feeding selectivity on speci?c prey. Unfortunately,the data in the present study cannot con?rm this fact be-cause Microcystis presented at very high densities (?106cells ml ?1) and predominated markedly in the phytoplank-ton community (average, 85%) during the bloom period in the pond. Hence, it is possible that the amoeba only utilized Microcystis because the latter was the most accessible and available prey or attachment substrate. We cannot exclude this possibility and therefore cannot conclude here whether the grazing on Microcystis is species speci?c or density dependent.

Most rhizopods are generally considered to associate with suspended particles or other substrates to feed on their prey (Rogerson and Laybourn-Parry 1992; Anderson and Rogerson 1995; Murzov and Caron 1996). Considering their feeding manner, it seems that Penardochlamys sp. also needs to attach to Microcystis colonies to feed effectively on the cyanobacterial cells (see Fig. 3). Thus, it can be consid-ered that the frequency of attachment to either Microcystis species strongly re?ects the prey availability for the amoeba within the two Microcystis species. In the present study, we found seasonal changes in attachment of the amoeba to colonies of two Microcystis species, indicating that the relative importance of these cyanobacteria as prey for the amoeba had also changed from M . aeruginosa to M .wesenbergii . Because the percentage of amoeba cells at-tached to M . wesenbergii colonies was positively correlated with the abundance of M . wesenbergii (Spearman’s r ?0.528, n ? 36, P ? 0.005), the shift could be explained by the seasonal changes in cell densities of both cyanobacterial species.

Previous laboratory studies reported that some rhizo-pods have a wide prey range within cyanobacterial species (Ho and Alexander 1974; Yamamoto and Suzuki 1984;Laybourn-Parry et al. 1987). In spring, prior to the bloom-ing of Microcystis , other cyanobacteria such as Phormidium mucicola and Anabaena sp. presented at high densities of 105–106 cells ml ?1 in Furuike Pond. If Penardochlamys sp.can utilize these cyanobacterial species as prey, their popu-lation should increase in spring, because the water tempera-ture and pH in spring were only slightly lower than those in fall (data not shown) when the maximum abundance of the amoeba was detected (Fig. 2B). However, Penardochlamys sp. was only detected when Microcystis spp. formed heavy blooms, and was rare in spring and winter when the abun-dance of Microcystis was low (Fig. 2). Thus, it is suggested

Fig. 3.A Penardochlamys sp. attached to a senescent colony of M .aeruginosa and B to a colony of M . wesenbergii , collected in Furuike Pond. All amoeba cells, indicated by arrows , have ingested Microcystis cells. Glutaraldehyde ?xation, bars 10μ

that the annual dynamics of the amoeba population are closely related with that of Microcystis, and food availability may be the primary reason for the restricted periods of amoeba occurrence in Furuike pond. Cook and Ahearn (1976) also reported that the naked amoeba Asterocaelum anabaenophilum, which fed speci?cally on Anabaena planktonica, was not detectable before the cyanobacterium bloomed.

Cook et al. (1974) reported that the abundance of a mayorellid-like amoeba rapidly increased following the bloom of A. planktonica, and that the grazing of the amoeba is a major cause of termination in the cyanobacterial bloom in Lake Sidney Lanier. In the present study, the abundance of Penardochlamys sp. changed markedly during the blooms of Microcystis spp. (Fig. 2), but was not signi?cantly related with Microcystis abundance (see Results). Thus, trophic interaction between Penardochlamys sp. and Microcystis spp. seems to be less important for determining the abundances of both these organisms in Furuike Pond, even though the former utilized only the latter as food. In addition, to evaluate the potential effect of grazing by Penardochlamys sp. on the Microcystis population, we roughly estimated the grazing impact during the study period from ingested cell numbers of Microcystis in food vacuoles of the amoeba (see Results) and the mean lifetime of a protistan food vacuole (20min, Fenchel 1987). Penardochlamys sp. consumed only 4.3% ?7.5% day?1 (mean ? SD) of the standing stock of the cyanobacterium from late May to November, and as a result, grazing by the testate amoeba may be of minor importance in controlling the abundance of the cyanobacterium during bloom periods.

We present here the seasonal abundance and unique feeding habits of Penardochlamys sp., and suggest that there is a trophic linkage between this testate amoeba and Microcystis spp. in Furuike Pond. Daft et al. (1985) and Dryden and Wright (1987) suggested that rhizopods are useful microorganisms for controlling cyanobacterial blooms through biological processes. Thus, our study may also provide information for evaluation of the effectiveness of rhizopod grazing for the biological control of cyano-bacterial blooms. Further quantitative studies are required to elucidate the trophic interactions between these protists and bloom-forming cyanobacteria in natural waters. Acknowledgments We are grateful to M. Hirose, M. Ueki, K. Murakami, and other members of the Division of Aquatic Biology and Ecology, Center for Marine Environmental Studies, Ehime University, for their support in the ?eld sampling. Thanks are also due to Dr. M. Morris for her correction of the English. In addition, Y. N. is grateful to the Laboratory of Marine Biodiversity, Graduate School of Fisheries Sciences, Hokkaido University, for providing logistical and ?nancial support for the data analysis and manuscript preparation. References

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