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Protistology 3 (1), 47-57 (2003)
Protistology
Optimizing methods of the recovery of gymn-amoebae from environmental samples: a test of ten popular enrichment media, with some observations on the development of cultures
Alexey V. Smirnov
Department of Invertebrate Zoology, Faculty of Biology and Soil Sciences, St.Petersburg State University, Russia
Summary
Enrichment cultures remain almost the only method of the recovery of gymnamoebae from the environment. The method depends on the media used and the choice of appropriate media that make it possible to recover the maximum number of species is of primary importance. Usage of many media and multiple examinations of dishes are very laborious and limit the involvement of amoebae in the ecological and faunistic studies of protists. The present study is aimed at the choice of the minimal set of media allowing for the recovery of appropriate fraction of amoebae fauna during the initial survey of a habitat. Two samples from Swiss localities were inoculated into 10 most widely used enrichment media. Results indicate that the most “productive” media in term of the number of recovered species are filtered water from the original habitat with wheat grains and cerophyl-Prescott agar. Number of inoculated dishes and quality of screening are more important than the range of media used. Enrichment cultures undergo a fast succession of species composition and at least two examinations of inoculates are required for reliable recovery of species diversity. Similarly to natural habitats, each enrichment culture contains a hidden community of amoebae. The paper offers a standard approach to the survey of amoebae biodiversity in freshwater benthos.
Key words: amoebae, species recovery, cultivation, ecology
Introduction
There are three ways to examine the biodiversity of protists in a local habitat: direct examination of fresh samples, enrichment cultivation of samples with subsequent examination of cultures, and the study of
total DNA extracted from the habitat. The first two approaches are classical, dating back to the beginning of protozoology, and allow a direct recovery of morphospecies. The latter method is relatively new and allows researchers to obtain sequences that may be further assigned to known morphospecies. Its evident advantages are the possibility to recover the DNA of
© 2003 by Russia, Protistology
non-culturable protists and a more precise estimation of the species number in a habitat (Moon-van der Staay et al., 2001; Lopez-Garcia et al., 2001). Study of the total DNA can show us how many species still remain non-recovered in a habitat. It also permits the estimation of their phylogenetic position and thus indicates a way towards species recovery (Dawson and Pace, 2002). For instance, one may use molecular data on previously overlooked taxa to choose the media, tools and methods that are likely to be suitable for their morphological recovery. However, only known sequences can be assigned to species and thus without the increment in the number of morphologically described, cultured and sequenced species this approach cannot progress. Thus, balanced development of the methods of morphological and molecular species recovery is important.
In some groups of protozoa, like gymnamoebae, direct examination of fresh samples is virtually useless and only enrichment cultivation allows the recovery of actual species diversity (Foissner, 1987; Arndt, 1993; Ronn et al., 1995). The heterogeneous structure of natural habitats (Fenchel, 1996; Anderson, 2001; Bischoff, 2002) results in the occurrence of numerous and diverse microhabitats harbouring a large variety of species and many of them require special efforts to be revealed (Fenchel et al., 1997). These cryptic organisms constitute a “seedbank” of species (Finlay et al., 1997), or “hidden community” (Smirnov, 2001). The fraction of hidden community that will be recovered by enrichment cultivation depends on the amount, schedule, and pattern of sampling and the variety of methods used to recover amoebae (Smirnov and Goodkov, 1995; Smirnov, 1999, 2001). In other words, the diversity of“observed community” is limited by our ability to create suitable conditions for the multiplication of as many species as possible.
The problem of more and less suitable enrichment media for isolation of gymnamoebae and other protists from the environment has long been discussed (Cutler, 1920; Severtsoff, 1922; Singh, 1946; Kyle and Noblet, 1986; Darbyshire et al., 1974, 1996; Menapace at al., 1975; Ronn et al., 1995). Page (1988) suggested six basic media for initial inoculation of freshwater samples, allowing the recovery of most amoebae species. This set is a difficult compromise between the trivial advice to use “as many diverse media as possible” and the realistic estimation of the painstaking job of examining multiple dishes. Usage of a greater number of enrichment media results in the increment of the list of recovered species, but practical needs demand to motivate a choice of a limited set of media allowing for the fast recovery of appropriately large fraction of amoebae fauna in a habitat. Another important
variable, influencing the results, is the complicated dynamics and pattern of amoebae development in multi-species cultures. This was noted early (Tsujitani, 1898; Cutler and Crump, 1927; Singh, 1942) but unfortunately this insight is now often ignored. Every scientist working with amoebae has a lot of empirical experience in the field of enrichment cultivations, but almost no comparative tests of different approaches have been performed (Foissner, 1987; Ronn et al., 1995). The present paper reports on the recovery of amoebae fauna from two samples of freshwater benthos using various media for inoculation and on some observation on the development of cultures. It offers methodological recommendations and suggests a standard approach to the initial survey of amoebae biodiversity in freshwater benthos.
Material and methods
Sampling. The upper 2-3 cm of the sandy bottom sediment was collected on May, 13, 2001 from the lake Neuchatel (Switzerland), and the upper 1 cm of detritus was collected from the neighboring shallow pond (Neuchatel Pond). Both samples were transported to the laboratory in termo-isolating containers and carefully mixed by gentle shaking. Immediately after shaking, 5 g of wet sediment from each sample were distributed into Petri dishes as described below.
Inoculation and media. A set of10 media (see Page, 1988) for recipes and abbreviations) was used to inoculate each sample. Five 90 mm Petri dishes per medium (with except for L medium, where three dishes were used) were inoculated, a total number of inoculated dishes was 48 per sample. Each dish received
0.1-0.2 ml of sediment. The media were:
1. NNA: non-nutrient agar without overlay (Page, 1988)
2. NNA+PJ: NNA with the overlay of Prescott-James medium (Prescott and James, 1955)
3. NNA+Cer: NNA with the overlay of 1% cerophyl infusion (Page, 1988)
4. CPA: Cerophyl-Prescott agar without overlay (Page, 1988)
5. CPA+PJ: CPA with the overlay of PJ medium
6. PJ: Prescott and James (1955) medium with wheat grain (one per dish)
7. Cer: 1% cerophyl infusion (Page, 1988) with wheat grain (one per dish)
8. SE: 0.05% soil extract (Page, 1988) with wheat grain (one per dish)
9. LW: filtered (Millipore 0.45 ^m) water from the Neuchatel lake with wheat grain (one per dish)
10. L: filtered (Millipore 0.45 ^m) water from the Neuchatel lake (3 dishes)
Negative control (non-inoculated media) accomplished the experiments. Wheat grains were autoclaved (10 minutes at 0.5 atm) before use.
Examination. Each dish was carefully examined twice (at 9-12 and 19-22 days of incubation) using inverted NikonDiaphot phase-contrast microscope. Each dish was screened under (100x) and (400x) magnifications until the repetitive observation of all found species (i.e. more than one specimen of each found species). At 19-22 days, all dishes with liquid media were examined under the dissection microscope for large amoebae. From the dishes containing agar media without overlay, all visible areas containing amoebae were cut of and transferred to 60 mm dishes with an identical agar medium and examined separately. The initial dishes were then covered with the overlay of PJ, left for several hours (to allow excystment of cyst-forming amoebae) and examined under the inverted microscope. One dish with each media was examined regularly with 1-2 days intervals starting from the inoculation, in order to trace the development of cultures.
Distinction and identification of rhizopods. Two groups of rhizopods were chosen for the study: Gymnamoebia and Himatismenida, both groups sensu Page (1987). For reliable species distinction, a provisional atlas of species found in inoculated cultures during the study has been composed using digital imaging system and each species was briefly characterized. Systematical species identification was out of scope of the study. Species were identified to the level of genus using light microscopy. The species name is given in the results only when the species has good distinctive features or if I was able to recognize it reliably. Species, designated as “sp.” are either new for science or cannot be identified without culturing and detailed studies.
Results
Biodiversity. A total of 30 different amoeboid organisms were recovered from the Neuchatel Lake sediments and 38 from the Neuchatel Pond sediments. Among them 16 species of Gymnamoebia and 4 of Himatismenida were found in Neuchatel lake sample, for Neuchatel Pond the number is 26 and 4, respectively (Tables 1-2).
Species occurrence and development of cultures. Species distribution across the dishes was found to be mosaic and showed a pronounced succession (Tables 1-2). Only 20% of the species from Neuchatel Lake and 30% from Neuchatel Pond produced stable populations which were found in the same dish at both examinations. Simultaneously, 15% of the species in
Neuchatel Lake sample and 30% of the species in Neuchatel pond sample were noted in both examinations, but not in the same dish (Table 3). These facts indicate that the culture in Petri dish evolves rapidly and that in most cases the community of amoebae in a single dish is unstable.
There is no clear correlation between the size of species and the time of finding. Present data do not allow the estimation of the precise number of amoeba, but the total number of findings of a species is a function of its abundance. From this consideration it is evident that the sample from Neuchatel Pond contains more amoebae that that from Neuchatel Lake (Tables 1-2). Table 4 shows that in the sample from Neuchatel Lake a nearly equal percentage of species occurred during the first and the second examination while in more amoebae-abundant samples from Neuchatel Pond most of the species were found during the first examination only. If we consider the recovery of individuals, the pattern is even more evident. The first examination recovered 9 species of amoebae from the Neuchatel lake sample and 24 from the Neuchatel Pond. The second examination increased these figures to 20 and 30 species, respectively. Table 3 also shows that in the sample from Neuchatel Lake, most of species (55%) were recovered during the second examination of samples while in the sample from Neuchatel Pond the corresponding number is only 15%. These data confirm that the succession is faster in denser cultures and the time of species finding depends more on this variable. However, dense cultures containing many species do not seem to be less stable because the overall percentage of repetitive findings is the same for both samples (Table 4).
After inoculation the evolution of a dish containing liquid medium is initiated by the rapid development of bacteria and in a few days the bottom of the dish gets covered with bacteria, diatoms, filamentous cyanobacteria and unicellular algae embedded in the biofilm. The fastest growth ofbacteria appears on the CPA+PJ, the slowest — in the LW or PJ media. After 3-4 days small ciliates, flagellates and other protists start to multiply and disperse through the dish. The first amoebae may be found to the 5-6th days, normally these are heteroloboseans and the smallest vannellids.
Further events depend on the medium and contingent events in the cultures. Generally after 8-10 days it is possible to see a community of small and medium-sized amoebae. At this stage large species nearly never occur, but few occasional findings are possible. It often happens that heteroloboseans become very abundant in culture. They explore the space rapidly and may feed on smaller amoebae. They can suppress the development of small species and such cultures will be dominated by one or two heterolobosean species
Notes: species, found only during the first examination of dishes are indicated with black rectangles, found only during the second examination - with mosaic rectangles, found during both examinations - with gray rectangles. Light gray shadowing of the species name indicated abundant species (frequency of findings >10%, i.e. > 5 dishes).
Cochliopodium sp
Notes: species, found only during the first examination of dishes are indicated with black rectangles, found only during the second examination - with mosaic rectangles, found during both examinations - with gray rectangles. Light gray shadowing of the species name indicated abundant species (frequency of findings >10%, i.e. > 5 dishes).
until encystment or death. However, they can never completely remove all other amoebae and when the feeding pressure of heteroloboseans decreases, other species start to develop in the dish.
For a given inoculum, the abundance and species composition of amoebae may be very different, depending on the success of amoebae and other biota in terms of the predation ofbacteria and destruction of the biofilm that covers the bottom of the dish. In some dishes (especially in rich, cerophyl-containing media) the biofilm is dense after 8-10 days, covering almost the entire area of the dish. Normally few amoebae may be found in such dishes; their motility is limited and they do not show typical locomotive morphology. It is very difficult to observe and identify species under such conditions and the risk of overlooking a species is high. But in most of the media amoebae are eventually able to clean several areas in the dish from the biofilm and they are perfectly visible in these areas. It seems that initial disruption of the biofilm occurs due to the activity of large and abundant predators such as the polyphagous amoebae species Vannella simplex or Cochliopodium spp., large ciliates and flagellates or even crustaceans, oligochaetes and occasionally other meiofauna.
The distribution of amoebae species across the bottom of the dish may be very heterogeneous. A species very often occurs only in certain areas of the dish. Some species strictly avoid nutrient-rich regions such as areas around wheat grains, while others preferably occur there. Certain species prefer to stay in the biofilm rather than in the clean areas of the dish. Many species, especially those belonging to the genera Vannella, Platyamoeba and Cochliopodium, form a “ribbon” of feeding amoebae at the border of clean area and the biofilm-covered area. Generally, the dish must always be thoroughly examined to recover all observable diversity of amoebae without special preference to the few clean areas.
During further development of cultures, smaller species and heteroloboseans start to encyst or die off, and after 20-21 days of incubation a community of large and medium-sized amoebae develops. Small species can still be found, but they are usually not numerous and appear only in few dishes. At this time a large part of the bottom of the dish is normally free from biofilm and amoebae are numerous and well-visible. Alternatively (if the medium is too nutrient-rich) the dish is totally covered with a thick biofilm and amoebae are few and hardly visible.
After 30-40 days no more amoebae can be recovered, and the bottom of the dish is covered with a layer ofbacteria, cyanobacteria, diatoms and algae. But in some dishes (mostly in those where the biofilm was
completely disrupted and removed) a culture may last up to 3-4 months, sometimes even longer.
Media. When analyzing the preferable media we must take into account the frequency of species findings (Fig. 1). Perhaps, species found in less that 10% of dishes (i.e., in 5 or less dishes) are rare in the habitat and their occurrence in the dishes depends mainly on their initial presence in the inoculum. The development of the remaining species probably depends mostly on the appropriate medium and on the conditions in the dish. When discussing more or less suitable media it is reasonable to consider only abundant species. Recovery of the remaining species appears to be a function of inoculate volume rather than of the medium used. The few species that only grow on agar media are an exception.
The most productive liquid medium for species recovery was in both cases LW (Table 5). It allowed for the recovery ofalmost all abundant species in the sample from Nechátel Lake, and 13 out of14 abundant species from Neuchátel Pond. The remaining species in this sample were recovered on CPA medium. If rare species are also considered the pattern is basically the same, but the total percentage of species recovery in LW decreases due to the increment of overall number of species.
Four species (Acanthamoeba sp., Dermamoeba sp., Thecamoeba sphaeronucleolus and Hartmannella vermiformis) were recovered only on the agar media and among two used media, CPA appears to be the most productive.
Cumulative charts indicating the accumulation of the species list during the study (Figs. 2-3) were empirically optimized to reach the saturation of the number of recovered species in the minimal number of media used. Both graphs show that LW was the most productive medium, followed with Cer and CPA or NNA. If rare species are also considered, the four listed media allow for the recovery of all species in the sample from Neuchátel Lake and 26 out of 30 species from Neuchátel Pond.
Discussion
Biodiversity. Altogether 20 gymnamoebae species from the Neuchátel Lake and 30 from the Neuchátel Pond were recovered. These numbers recovered from a single sample are comparable or even exceed those reported in several previous faunistic studies of gymnamoebae (Sawyer, 1980; Garstezki and Arndt, 2000). However, detailed long-term studies of local habitats always recover more species of rhizopods (Anderson, 1997; Butler and Rogerson, 2000, Finlay et al., 2000). It seems logical to suggest that the relative abundance of various microniches is somehow related
Table 3. Species recovery during the study (percent of recovered species from the total number of species).
Site First examination only Second examination only Found in the same dish in both examinations Found in both examinations, but not in the same dish
Neuchâtel Lake 10% 55% 20% 15%
Neuchâtel Pond 20% 20% 30% 30%
with the global environmental conditions, and thus can vary during different seasons. Chances to recover rare species are higher in long-term studies due to the larger number and volume of treated samples.
Species occurrence and development of cultures. Mosaic species distribution among dishes may be explained either by their initial heterogeneous distribution in inoculated sample or by the differences in the species development in cultures. Probably both variables contribute.
The smallest aggregates of detritus can not be dispersed by shaking, without application of a mechanical homogenizer (which will damage amoebae cells and is thus non-acceptable). There is increasing evidence for the presence of microhabitats in the environment and for patched distribution of amoebae (Smirnov et al., 1998; Smirnov, 2002; Anderson, 2001; Bischoff, 2002). Certain amoebae species probably
exist in patches in connection with physical aggregates of detritus or other material. These aggregates probably can not be dispersed into single cells even after heavy shaking. On the other hand, certain species (especially medium-sized and large amoebae) are evidently rare in the environment and thus appear in only a minor percentage of dishes and show random distribution.
Mixed cultures show a rapid succession of species. Once more it confirms the idea of the presence of a high number of very diverse microhabitats that are selectively occupied in the environment as well as in the Petri dishes. It is especially remarkable that all dishes, even with the same medium, look different from one another, showing the development of specific conditions and specific protozoan communities. Perhaps a limited volume of medium, random selection of inoculated species and lower diversity of the food supplies than in nature, results in the development of a
Fig. 1. Number of species findings (total of 48 dishes) in the samples from Neuchâtel Pond (left) and Neuchâtel Lake (right).
Table 4. Overall finding of species during the study (percent of findings from the total number of amoebae findings).
non-stable community that undergoes rapid species succession.
On the other hand, observed successions in the Petri dishes indicate that many species that are actually present in the dish may remain unobserved even under the most careful examination. In other words, all dishes contain a “hidden” community of species that produce at least two rather different “observed” communities during the period of study. Perhaps this also takes place in the environment. The composition of sufficiently abundant species that are actually recovered during the study may undergo drastic changes due to the variation in the composition and the quantitative ratio of microhabitats each of which harbors a certain set of species. It may produce the impression of successional changes and dynamics of species composition. But the actual species composition (hidden community) may be constant.
Dishes containing few species of amoebae show a much more stable development of the cultures and slower succession changes than do species-rich dishes. This suggests that the food competition and mutual predation of amoebae may be an important factor determining the dynamics of the species occurrence. Many large and medium-sized amoebae are
polyphagous and feed on smaller amoebae of other species and even smaller individuals of their own species (Page, 1988; Smirnov, 1999).
The present results show that the development of enrichment cultures is complicated and apparently unpredictable. The “ecology of a single Petri dish” requires careful attention, especially when the data are intended for comparative analysis. One must always keep in mind that each inoculated dish constitutes a unique habitat and several dishes with the same media may develop different communities. Actually a set of dishes with identical medium does not represent a replica, but they do increase our chances to recover more species from the habitat and thus to obtain a more reliable estimate of the actual species diversity.
Media. The results reported in the present paper accord with my previous experience in amoebae species recovery from freshwater and marine sediments (Smirnov and Goodkov, 1995; Smirnov et al., 1998; Smirnov, 1999, 2001). It is possible to recommend the following approach to the initial survey of amoebae biodiversity in freshwater benthos:
Media: at least one liquid medium and one agar medium. Based on the present observations I suggest the following for freshwater samples:
Table 5. Percent of the recovered species (100 % — overall number of species in the location) for each medium (for media abbreviations see Material and methods).
Sites Media LW PJ Cer SE NN +PJ CPA +PJ CPA NNA +Cer NNA L
Neuchâtel Frequent species 100% 89% 78% 78% 45% 56% 22% 22% 56% 78%
Lake All species 65% 60% 55% 40% 20% 40% 15% 15% 40% 45%
Neuchâtel Pond Frequent species 88% 76% 70% 65% 76% 53% 47% 65% 59% 18%
All species 60% 60% 53% 37% 57% 40% 40% 40% 40% 10%
Fig. 2. Cumulation of the number of recovered amoebae species from the Neuchatel Lake sediments. Chart is optimized to reach saturation in the minimal number of steps.
Fig. 3. Cumulation of the number of recovered amoebae species from the Neuchatel Pond sediments. Chart is optimized to reach saturation in the minimal number of steps.
1. Millipore-filtered water from original habitat with wheat grains
2. CPA
Filtered water from original habitat may be replaced with the cerophyl infusion or PJ medium with wheat grains. Cerophyl appears no longer to be commercially available, but dried cereal leaves may be an appropriate replacement. However, it seems logical to suggest that the water of original habitat that has a characteristic chemical composition may favour the creation of more appropriate michoniches. It results in the development of more species than artificial, chemically-defined media.
Inoculation: not more than 0.1-0.2 ml of the sample per 90 mm dish; only one wheat grain per dish. Patches of the inoculated material must leave enough free bottom space to observe amoebae, and bacterial and fungal growth in the dish must not suppress the amoebae. The number of inoculated dishes is of primary importance, and the increment in the number of dishes seems to be more important than the usage of extra media.
Observation: Inverted phase-contrast microscope; magnifications 100x and 400x. Inoculated dishes kept under usual room temperature (20-22oC) must be examined at least twice, at 9-12 and 20-21 days of incubation to record both small and medium-sized species; in case of other temperature of maintenance this time must be respectively corrected. During the second examination it is desirable to screen dishes under dissection microscope to seek for few specimens of the
largest amoebae that may develop by this time. Dishes with CPA should be screened initially under dissection microscope after 9-12 days of incubation. Material from suspected amoebae-containing areas is to be transferred to the microscope slides and examined by routine microscopy. The dish should preferably be covered with PJ medium and examined using low magnification of the inverted microscope after 20-21 days.
The above approach allows the recovery of the basic fauna, presumably most abundant and actively participating in the mass and energy flows at the moment of sampling. It may be sufficient for initial survey and ecological purposes. For the detailed study of the biodiversity, further increment in the number of recovered species may be reached by usage of extra enrichment media mentioned here and described in the literature (see citations in the text). Everybody working with the biodiversity of amoebae should keep in mind that the temporal and spatial pattern of amoebae occurrence is very complex, and only multiple sampling of a good variety of ecotopes within a habitat done under different conditions (seasons, weather, etc.) allows for the more or less reliable recovery of amoebae fauna.
Acknowledgements
The present work was financed by SNSF grant 7SUP J062343 and INTAS YSF 2001/2-0048 grant. I am most thankful to Jan Pawlowski for hospitality, sampling trips and providing facilities for this study and
to Maria Holzmann and Elena Nassonova for help in sampling and interesting discussions. Special thanks are due to Tom Fenchel and Bland Finlay, whose ideas on the cryptic diversity, species recovery and global distribution of protists in the biosphere led me to perform this experiment. Both contributed greatly to this paper by good criticism of ideas and fruitful discussions of the manuscript. Tom Fenchel kindly did a great work on editing and clarification of the text of the present paper.
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Address for correspondence: Alexey V. Smirnov. Dept. of Invertebrate Zoology, Faculty of Biology and Soil Sciences, St. Petersburg State University, Universitetskaja nab. 7/9. 199034 St. Petersburg, Russia. E-mail: smirnov@home.rclph.spbu.ru.
The manuscript is presented by A.V. Goodkov
