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Two new species of marine amoebae: Hartmannella lobifera n. sp. and Korotnevella nivo n. sp. (Lobosea, Gymnamoebida)
Arch. Protistenkd. 147 (1996/97): 283-292 © by Gustav Fischer Verlag
ARCHIV
FUR
PROTISTEN KUNDE
Two New Species of Marine Amoebae: Hartmannella lobifera n. sp. and Korotnevella nivo n. sp. (Lobosea, Gymnamoebida) ALEXEY
V.
SMIRNOV
Marine Biological Laboratory Helsing0r, University of Copenhagen, Denmark; Department of Invertebrate Zoology, Faculty of Biology & Soil Sci., St. Petersburg State University, St. Petersburg, Russia
Summary: Two new species of marine amoebae, isolated from the Sound (Denmark) are
described. Comparison of Korotnevella nivo n. sp. with other known species of this genus indicated that it is difficult to use the structure of surface scales in Paramoebidae as a taxonomic character. One of the characteristic features of Hartmannella lobifera n. sp. is its remarkable cyst structure.
Key Words: Amoeba; Gymnamoebia; Paramoebidae; Hartmannellidae; Ultrastructure.
Introduction The number of named species of marine amoebae is still relatively low, compared with that of freshwater and soil species (PAGE 1983a, 1991). Some workers have studied marine amoebae without species identification or detailed descriptions (PENNICK & GOODFELLOW 1975; ANDERSON 1977), and so the presence of the members of some genera in salt water is known with certainty whereas very few or no forms are described at the specific level (PAGE 1983a). Only one marine species of Hartmannella is known (PAGE 1980) and no taxonomically valid descriptions of marine species of Dactylamoeba exist. Two representatives of these genera, isolated from the brackish-water bay (salinity about 15%0) are described in the present paper. The application of the generic name Dactylamoeba KOROTNEFF, 1880 proposed by PAGE (1982, 1983a, 1983b) was found to be taxonomically incorrect (GOODKOY 1988). One reason is that the generic name Dactylamoeba is already included by the International Commission of Zoological Nomenclature in the list of generic names with the remark that it is a synonym of the generic name Mayorella (PAGE 1982). Another rea-
son is that PAGE (l983a), when he proposed the generic name Dactylamoeba, suggested that the type species of the genus - D. elongata KOROTNEFF, 1880 - possesses microsales. This could be checked only with electron microscopy, however, this species was not studied with EM. Moreover, the description of D. elongata is insufficient: it does not contain any information which allows reasonable re-isolation of this species in future. Thus, the use of the name Dactylamoeba as the name of the nominal genus is incorrect, and the name Korotnevella, which is used in this paper, has been proposed as a substitute (see GOODKOV 1988 for details).
Material and Methods The amoebae were isolated from the superficial 1-1.5 mm of sediments from Niva Bay (Sound; 15 km South of Helsing0r, Denmark). Samples of sand with patches of white sulphur bacteria were collected and inoculated in either 90 mm Petri dishes with seawater overlay (salinity about 35%0) and two wheat grains in each dish, or on C75S agar
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(PAGE 1983a) with seawater overlay. Both species were able to live on C100S agar with overlay (op. cit.), but they multiplied more slowly then on C75S agar. The amoebae were observed and photographed adhering to the glass surface. Permanent preparations, stained with iron haematoxylin and with Kernechtrot were made as described by PAGE (1988). Feulgen staining was performed as described by LEE & PAWLOWSKI (1992). For electron microscopy, amoebae were fixed as follows: (1) in a mixture of equal volumes of 4% buffered glutaraldehyde, 2% osmium tetroxide and 0.1 M phosphate buffer (pH 7.4) for 40 min; (2) in buffered 4% glutaraldehyde with sucrose (10 mg/ml) at pH 7.4 for 30 min, followed by a rinse in buffer and postfixation with 2% osmium tetroxide for 2 hours; (3) in 2% buffered osmium tetroxide (pH 7.4) for 30 min. In all cases the amoebae were washed 3 times in the buffer after fixation. After dehydration with an ethanol series specimens were embedded in Epon. Sections were stained with a saturated solution of uranyl acetate dissolved in 70% ethanol and with Reynolds lead citrate and examined with a Zeiss EM 900 electron microscope.
Results Hartmannella lobifera n. sp. Locomotive amoebae (Figs. 1-4) were always monopodial, with an anterior hyaline cap which from time to time was obliterated by intruding granules. When the amoeba starts to move the cap may be somewhat narrower than the rest of the cell. The body of the locomotive form was usually equally wide at its anterior, posterior and middle parts. Amoebae moved by steady flowing of the cytoplasm; no evidence of eruption was seen. Seventy specimens were measured on the glass surface. The length of the locomotive form varied form 28 to 42 11m (mean 35.2 11m) and the breadth from 5 to 8 11m (mean 6.8 11m). Lengthlbreadth ratio (LIB) varied between 4 and 7 (mean 5.1). The presence of 1-3 small posterior hyaline lobes was very common (Figs. 3-4). The formation of these lobes was a very characteristic process connected with the changing of the direction of movement (see below). Some specimens in locomotion had no lobes, but instead they had a broad, not very well differentiated morulate uroid (Fig. 1) and some had no differentiated uroid at all. Villous uroid or trailing uroidal filaments were never observed. The amoebae frequently changed direction of locomotion. In fact it was difficult to find a monopodial locomotive form, because most amoebae produced pseudopodia, often in two or three different directions simultaneously (Figs. 7-8,10). The amoeba can change the direction of locomotion by turning its anterior end, but more often it produces a lateral pseudopodium. This
pseudopodium becomes the leading one and the amoeba starts to move in a new direction. While the major part of the amoeba body progresses in the new direction the remnants of the previous hyaline can remain at the same place. Thus it moves along the amoeba body towards its posterior end (Figs. 5-6). When it reaches the end of the cell it separates from the glass and· forms a posterior hyaline lobe. When not moving ("resting") the amoebae were generally rounded, often with 3-5 distinct hyaline lobes (Fig. 9). The amoebae did not produce a differentiated floating form; floating amoebae were often irregular and produced pseudopodia in all directions. A vesicular nucleus was discernible in both live (Fig. 13) and in stained (Fig. 14) specimens. Its diameter as measured in stained preparations, was 3.5-6 11m and the diameter of the nucleolus was I-211m. A small contractile vacuole was usually situated in the posterior part of the body (Figs. 5-6), but sometimes it was observed moving within the cell (Fig. 2). Prior to systole it was always situated near the surface at the posterior part of the cell. The refractile spheres, about 1 11m in diameter, were the most characteristic cytoplasmic inclusions. Their number varied from 3 to 42 per cell. The cytoplasm also contained many small opaque granules and some food vacuoles. After 3-4 weeks of cultivation on medium (2) the amoebae produced cysts (Figs. 11-12). The cysts had two distinct walls: a thin outer wall and a thicker inner one. The cell was sealed to the inner wall of the cyst. The slightly ovoid endocyst was always situated eccentrically inside the spherical ectocyst, being attached to the outer wall from one side and leaving a distinct empty space on the other side. No cyst pores were detected. The diameter of the ectocyst was 10-12 11m and the maximum dimension of the endocyst was 8-11 11m. All observed cysts were uninucleate. The fine structure of this species was found to be typical of Hartmannellids. The nucleus (Figs. 15-16) was spherical, with a central and slightly heterogeneous nucleolus (Fig. 16) and without any differentiated inner nuclear lamina. The cell surface coat (Fig. 17) consisted of a thin (about 20 nm) glycocalyx without any visible differentiation. The mitochondria (Fig. 18) were rounded or elongate, with tubular cristae. Dictyosomes (Fig. 19) were few and consisted of 3-6 flattened cisternae. Some membrane-bounded inclusions (Fig. 20) with a rounded or irregular form, were present in sections. Fig. 21 shows the contractile vacuole, which was situated in the posterior part of the body, near the cell surface. It was surrounded by regularly arranged microfilaments and by small vesicles; several mitochondria were always situated around it. In all studied cells the granuloplasm was separated from the hyaloplasm by a layer of microfilaments (Fig. 22).
Hartmannella lobifera n. sp. and Korotnevella nivo n. sp.
Microfilaments were also abundant in the granuloplasm and sometimes they were arranged in short cores, but careful searching did not indicate the presence of cytoplasmic microtubules.
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A study of the cysts (Figs. 23-26) indicated the presence of three amorphous distinct layers in the endocyst. The total thickness of the endocyst wall was about 170 nm. The ectocyst was finer and its wall appeared to
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Figs. 1-14. Hartmannella lobifera: 1-4 - in locomotion; 5-6 - formation of the posterior hyaline lobe; 7-8 - the cell during non-directed movement; 9 - when not moving; 10 - usual view of amoebae on the glass surface; 11 - cyst; 12 - empty cyst; 13 - nucleus in living cell; 14 - nucleus in stained preparation. (Abbreviations: iw - inner cyst wall; 1- hyaline lobe moving along the cell; n - nucleus; ow - outer cyst wall; pl- posterior hyaline lobes; u - uroid.) Scale bar 10 11m throughout (note lower magnification in Fig. 10).
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Figs. 15-22. Hartmannella lobifera: 15 - total section of the cell; 16 - nucleus; 17 - cell surface; 18 - mitochondria; 19dictyosome; 20 - microbody-like structure; 21 - contractile vacuole; 22 - microfilaments at the border of the granuloplasm and the hyaloplasm. (Abbreviations: cv - contractile vacuole; m - mitochondria; mf - microfilaments.) Scale bars: Figs. 17 and 19 - 0.25 11m; others - 0.5 11m.
Hartmannella lobi/era n. sp. and Korotllevella Ilivo n. sp.
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Figs. 23-26. Hartmannella lobifera: 23 - total section of the cyst; 24 - cyst wall in cross section; 25 - gutters in saggital section; 26 - gutters in cross section. Figs. 27 and 28. Korotnevella nivo: 27 - nucleus; 28 - scales. (Abbreviations: c cytoplasm; eb - electron-dense body; em - patches of electron-dense material; et - electron transparent zone; g - channels; is - internal space of the cyst; iw - inner cyst wall; ow - outer cyst wall.) Scale bar 0.5 ~m throughout.
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have a filamentous nature. Its thickness was about 160 nm. The endocyst was always situated eccentrically within the ectocyst (Fig. 23) and a distinct eccentric empty space between them was always observed. Within the agar, ectocysts of neighbouring cysts may be closely adjacent one to another (Fig. 26). The surface of the cell within the cyst had channels (Figs. 25-26) which formed invaginations of the cell surface. These channels were approximately parallel at neighbouring sites on the surface, but could have different orientations in different regions of the surface (Fig. 25). The material of the inner cyst wall penetrated some of these channels (Fig. 26), while the others were empty in sections. Their functional significance remains unknown.
Korotnevella nivo n. sp. During locomotion (Figs. 29-33) the amoebae had a variable shape; usually they were elongate or more or less triangular. They produced only hyaline subpseudopodia (Figs. 29-32) which were short and finger-shaped or long (up to 1.5 times the body length) and conical. The latter type is very characteristic for this species. The cell could sometimes be broad and triangular, with 3-5 anteriorly directed, long pseudopodia. In this case it resembled some marine members of the genus Vexillifer (PAGE 1983a). The locomotive form without long pseudopodia resembled that of Neoparamoeba pemaquidensis (PAGE 1970, 1983a; as Paramoeba) or the limnic Korotnevella stella (see PAGE 1991). The frontal hyaloplasm always formed the border which had a variable width (Figs. 29-30) and which was more flattened than the granuloplasmic region of the body. The pseudopodia often continued as ridges on the dorsal surface of the amoeba (Fig. 31) and three or four of these ridges were usually present in the locomotive form. In most cases there was no differentiated uroid, but sometimes the posterior end of the amoeba was plicate or irregularly morulate (Fig. 33). During continuous locomotion the amoebae always lacked differentiated uroidal structures. Eighty-five live specimens were measured in the culture dishes with the use of a water immersion objective. The length of the locomotive form varied between 19 11m and 51 11m (mean 39 11m); the breadth varied between 14 11m and 40 11m (mean 21 11m). Mean lengthlbreadth ratio (LIB) was l.9 (range: 0.95-2.8). The subpseudopodia never determined the direction of locomotion. In order to change the direction of movement the amoeba formed a broad wave of hyaloplasm
which was thrown out in the current direction of movement. When resting the amoeba had an irregular shape (Fig. 34) or rounded (Fig. 35) with many short, hyaline finger-shaped projections. These projections were formed in all directions from both the lateral and the dorsal hyaloplasm. The floating form (Fig. 36) was radial and it had from three to fifteen long, tapering and pointed hyaline pseudopodia. The longest were 3-4 times the diameter of the central mass of cytoplasm. The nucleus was vesicular with a single nucleolus which was located centrally or slightly eccentrically. The nucleolus was visible in both living specimens (Fig. 37) and in haematoxylin-stained preparations (Fig. 38). The diameter of the nucleus, measured in living specimens, was 2.2-3.8 11m, that of the nucleolus was l.2-2.2 11m. In stained preparations the nucleus was 3.4-4.5 11m in diameter. A para some was not detected, neither in living specimens (Fig. 37) nor in stained preparations. Feulgenstained preparations (Fig. 39) showed a slightly Feulgenpositive nucleus with a small central or eccentric Feulgen-negative lacuna and no evidence of a parasome. DAPI staining (Fig. 40) showed a homogeneously fluorescent nucleus without discernible internal differentiation and no evidence of other fluorescent inclusions, except for the food vacuoles in some specimens. No contractile vacuole was observed. The cytoplasm was packed with different types of inclusions including small food vacuoles, optically opaque granules of different size and shape, but no crystals were detected. Cysts were not found in the cultures. Electron-microscopic examination showed that the cell coat of this species bore scales (Fig. 28). They were generally basked-shaped and looked very like the scales of Paramoeba eilhardi illustrated by GRELL & BENWITZ (1970). The length of each scale was 400-514 nm, the breadth 170-230 nm and the width about 200 nm. The nucleus (Fig. 27) had a very distinct structure. At its middle, or somewhat eccentric, a compact rounded mass of electron-dense material was situated measuring about 1.6 11m in diameter. It was surrounded by an area of less electron-dense material which formed an irregular, but generally rounded body in the center of the nucleus. In some specimens this body made direct contact with the nuclear envelope, but often there was a distinct space between the body and the nuclear envelope which was filled with patches of electron-dense material. A differentiation of the fibrillar component in the nucleus was not observed; there was no nuclear lamina. With respect to its dimensions and position, the dense central body
Figs. 29-40. Korotnevella nivo: 29-30 - in locomotion; 31 - showing dorsal ridges; 32 - with long anterior pseudopodia; 33 - without these pseudopodia; 34 - in non-directed movement; 35 - when not moving; 36 - floating form; 37 - nucleus in living specimen; 38 - haemotoxylin-stained preparation; 39 - Feulgen-stained preparation; 40 - DAPI-stained preparation. (n - nucleus.) Scale bar 10 11m throughout.
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seems to correspond to the Feulgen-negative lacuna (Fig. 39), while the less dense material surrounding it corresponds to the "endosome" which was visible in haematoxylin-stained preparations (Fig. 38). No paras orne was detected at the electron-microscopical level during the examination of serially-sectioned amoebae. A satisfactory fixation of the cytoplasm and of the organelles was not obtained, but it was possible to conclude that the mitochondria of this species had tubular cristae and an electron-dense matrix. The single large dictyosome was always situated near the nucleus. Rounded lipid bodies were common in the cytoplasm.
Systematic account Hartmannella lobifera n. sp.
This "limax" (PAGE 1981) amoeba, moving by steady flow of the cytoplasm, with tubular mitochondrial cristae and a vesicular nucleus can undoubtedly be classified as belonging to the family Hartmannellidae (VOLKONSKY, 1931) PAGE, 1974. Within this family it cannot be assigned to the genus Glaeseria VOLKONSKY, 1931 due to the absence of nuclear division in cysts. There is no evidence of the villous-bulbous uroid and it always has a hyaline cap during locomotion. These two important features distinguish the present species from members of the genus Saccamoeba (FRENZEL, 1892) BOVEE, 1972. Any relationship between this species and the only well-studied member of the genus Cashia PAGE, 1974 - C. limacoides - is questionable. The frontal hyaline cap is usually well pronounced in the present species, whereas in C. limacoides it "might be completely absent (frequently) or present as a small hyaline tip or crescent" (PAGE 1974, p. 169). The formation of a bulbous uroid is noted for C. limacoides, while in the species described in this paper the uroid, if present, is morulate. Often the amoeba has one or two posterior hyaline lobes or no differentiated uroidal structures at all. PAGE (1986, 1988) noted, that in C. limaco ides, mitochondria are never elongate and have helical cristae, while in the present species they are often elongate and show no evidence of helical cristae. The most appropriate generic home for the species described above, is the genus Hartmannella (ALEXIEFF, 1912) PAGE, 1974. The species is similar to the known representatives of this genus with respect to locomotive morphology, formation of the lateral pseudopodia while changing direction of movement and absence of a villous-bulbous uroid. The posterior hyaline lobes which are common for this species were also noted for H. cantabrigiensis (PAGE 1974, Fig. 43). The fine structure of the present species corresponds well to that known
from studied freshwater and marine species of Hartmannella (PAGE 1980,1986). Cup-like structures or any other discrete elements in the glycocalyx of the present species were not found, but they were possibly destroyed during fixation. In the marine H. abertawensis, differentiated elements in the glycocalyx, were also not observed with certainity (PAGE 1980). The present species differs from all known species of Hartmannella due to its locomotive morphology and the cyst structure. These features warrant a description of a new species - Hartmannella lobifera. Diagnosis: Hartmannella lobifera n. sp. Hyaline cap sometimes obliterated by the intruding granuloplasm. Posterior hyaline lobes common, sometimes a morulate-like uroid is present. Length of the locomotive form varies from 28 to 42 /lm (mean 35.2 /lm); breadth varies from 5 to 8 /lm (mean 6.8 /lm). Lengthlbreadth ratio (LIB) varies from 4 to 7 (mean 5.1). Nucleus 3.5-6 /lm in diameter, nucleolus 1-2 /lm. Contractile vacuole present when amoebae are cultivated in 20%0 seawater. Capable of living and multiplying in 35%0 seawater and does not survive in freshwater. No cytoplasmic crystals. Cysts 10-12 /lm in diameter. Endocyst always situated eccentrically, being attached to the ectocyst at one side and separated from the latter by a distinct empty space at the opposite side. Inner cyst wall consists of three amorphous layers; its thickness is about 170 nm. Outer wall appears to have a fibrous nature; its thickness is about 160 nm. Known habitat: The Sound, Niva Bay (brackish-water, salinity about 15%0), Denmark. Type slides are deposited at the British Natural History Museum (London, U.K.). Holotype: 1995:9:6:5; Paratype: 1995:9:6:6. Differential diagnosis: The present species is three times larger then the only known marine species of Hartmannella, H. abertawensis; it has a different uroid and a different cyst structure. The latter feature also differentiates it from any described freshwater member of this genus. Korotnevella nivo n. sp.
The combination of light- and electron-microscopical features of this species (finger-shaped hyaline pseudopodia ["dacty10podia"], absence of the parasome, radiate floating form, microsca1es) gives good reason to classify it as a member of the genus Korotnevella (PAGE, 1981) GOODKOV, 1988 (Gymnamoebia, Paramoebidae). Within this genus only two freshwater species - K. bulla and K. stella - have been studied in detail including the use of electron microscopy (PENNICK & GOODFELLOW 1975; PAGE 1981). Both are clearly distinct from the present species with respect to light-microscopical morphology. Possibly, electron-microscopic study of some
Hartmannella lobifera n. sp. and Korotnevella nivo n. sp.
strains of Mayorella sensu lato, which are described only from light-microscopic data (BOVEE & SAWYER 1979; PAGE 1983a, 1991 and references therein), will show them to be scale-bearing. PENNICK & GOODFELLOW (1975) illustrated (using TEM) two unidentified marine scale-bearing amoebae (designated as Mayorella sp. 1 and Mayorella sp. 2). The first species has scales which are clearly distinct from those of the present species. There are some similarities between the scales of the present species and those of "Mayorella sp. 2", but the absence of any lightmicroscopic descriptions of these species leaves no possibility for further comparison. An unidentified parasome-free amoeba strain studied by GRELL & BENWITZ (1970) has scales closely resembling those of the present species (and of P. eilhardi). But in the case of this species, the authors (op. cit.) stressed a close morphological similarity to P. eilhardi while the present species is much smaller and has different pseudopodia. An unidentified strain studied by ANDERSON (1977), is more similar to the present species in dimensions and has scales of the same shape, though smaller than those of the present species. The pattern of nucleolar organisation, illustrated by ANDERSON (1977, p. 371) for his strain is different to that described here, but the absence of a light-microscopical description of ANDERSON'S strain does not allow for a more detailed comparison. The above discussion warrants the description of a new species - Korotnevella nivo (the specific name corresponds to the Danish pronunciation of the name of the bay where the species was found). Comparison of the information given by ANDERSON (1977) and GRELL & BENWITZ (1970) with the present description suggests that at least three different species of marine Korotnevella exist (given that the strain, studied by GRELL & BENWITZ is not a parasome-free strain of P. eilhardi). They all seem to show morphological differences, but all have the same general pattern of scales. Scales of P. eilhardi also closely resemble scales of these species. In contrast, two freshwater species, K. stella and K. bulla, each has a distinct scale pattern. Recently a freshwater Korotnevella was isolated and studied (SMIRNOV, unpublished). It has discoid scales with a conical projection in the middle of each scale. Such scales are different from those of any described species, but they closely resemble those of "Mayorella sp. 3" (PENNICK & GOODFELLOW 1975, Plate 5, Fig. e). This indicates that the taxonomic importance of such features as shape and dimensions of the scales in amoebae needs further detailed study. Diagnosis: Korotnevella nivo n. sp. Body triangular or elongate during locomotion. Subpseudopodia hyaline, short finger-shaped or long conical (up to 1.5 times body length). Mean length of the
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locomotive form 39 f.lm (19-51 f.lm), mean breadth 21 f.lm (14-40 f.lm). Mean lengthlbreadth ratio (LIB) 1.9 ranging between 0.95 and 2.8. Nuclear diameter 2.2-3.8 f.lm in live specimens and 3.4-4.5 f.lm in stained preparations. Capable of living and multiplying in seawater (35%0) and does not survive in freshwater. Length of each scale 400-514 nm, breadth 170-230 nm and width about 200 nm Known habitat: The Sound, Niva Bay (brackish-water, salinity about 15%0), Denmark. Type slides are deposited at the British Natural History Museum (London, U.K.). Holotype: 1995:2:9:3, Paratype: 1995:2.9:4. This isolate is deposited with the CCAP No. 1543/1. Differential diagnosis: Korotnevella nivo differs well from both freshwater species of this genus by the tendency to produce very long anterior hyaline pseudopodia. It does not survive in freshwater. It has a nucleus with a distinct structure which differs from that of any freshwater scale-bearing species. At the light-microscopic level it may be confused with some marine Vexillifera, but the structure of the cell coat allows it to be differentiated from the members of this genus.
Acknowledgements: This work was carried out during my stay at the Marine Biological Laboratory, University of Copenhagen, supported by a Danish Government Scholarship. I am most grateful to Prof. T. FENCHEL for providing the laboratory facilities and for helpful discussions on the paper. N. V. LENTS MAN helped me greatly during preparation of the text; Dr. A. B. GOODKOV made valuable comments to the manuscript. The work was supported with the Danish Natural Science Research Grant 11-0088-1 provided to Prof. T. FENCHEL.
References ANDERSON, O. R. (1977): Fine structure of a marine ameba associated with a blue-green alga in the Sargasso Sea. J. Protozoo!. 24: 370-376. GOODKOV, A. V. (1988): Korotnevella nom. nov. - new generic name for scale-bearing Mayorella-like amoebae. Zoo!. Zmn. 67: 1728-1730 (in Russian). GRELL, K. G. & BENWITZ, G. (1970): Ultrastruktur mariner Amoeben. I. Paramoeba eilhardi SCHAUDlNN. Arch. Protistenkd. 112: 119-137. LEE, J. J. & PAWLOWSKI, I. (1992): Feulgen staining the nuclei offoraminifera. In: LEE, 1. 1. & SOLDO, F. 1. (eds.), Protocols in Protozoology. Soc. of Protozoologists, Kansas, C l2-J-C 12-1. PAGE, F. C. (1970): Two new species of Paramoeba from Maine. 1. Protozool. 17: 421-427. - (1974): A further study of taxonomic criteria for limax amoebae, with descriptions of new species and a key to genera. Arch. Protistenkd. 116: 149-184. - (1980): A light- and electron-microscopical comparison of limax and flabellate marine amoebae belonging to four genera. Protistologica 16: 57-78.
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- (1981): Mayorella SCHAEFFER, 1926 and Hollandella n. g. (Gymnamoebia), distinguished by surface structure and other characters with comparison of three species. Protistologica 17: 543-562. - (1982): Mayorella SCHAEFFER, 1926 (Rhizopoda: Amoebida): proposed conservation. Bull. Zool. Nom. 39: 214-217. - (1983a): Marine gymnamoebae. Inst. Terr. Ecol., Cambridge. - (1983b): Three freshwater species of Mayorella (Amoebida) with a cuticle. Arch. Protistenkd. 127: 203-233. - (1986): The limax amoebae: comparative fine structure of the Hartmannellidae (Lobo sea) and further comparisons with the Vahlkampfiidae (Heterolobosea). Protisto logic a 21: 361-383. - (1988): A new key to freshwater and soil gymnamoebae. Freshwater BioI. Ass., Ambleside, U. K.
- (1991): Nackte Rhizopoda. Protozoenfauna. Bd. 2. Stuttgart, New York. PENNICK, N. C. & GOODFELLOW, L. P. (1975): Some observations on the cell surface structures of species of Mayorella and Paramoeba. Arch. Protistenkd. 117: 41-46. Accepted: August 21, 1996 Published: April 1997
Author's address: ALEXEY V. SMIRNOV, Department of Invertebrate Zoology, Faculty of Biology & Soil Sci., St. Petersburg State University, Universitetskaja nab. 7/9, 199034, St. Petersburg, Russia. FAX: +7 812 2180852; e-mail: [email protected]
ARCHIV
FUR
PROTISTEN KUNDE
Two New Species of Marine Amoebae: Hartmannella lobifera n. sp. and Korotnevella nivo n. sp. (Lobosea, Gymnamoebida) ALEXEY
V.
SMIRNOV
Marine Biological Laboratory Helsing0r, University of Copenhagen, Denmark; Department of Invertebrate Zoology, Faculty of Biology & Soil Sci., St. Petersburg State University, St. Petersburg, Russia
Summary: Two new species of marine amoebae, isolated from the Sound (Denmark) are
described. Comparison of Korotnevella nivo n. sp. with other known species of this genus indicated that it is difficult to use the structure of surface scales in Paramoebidae as a taxonomic character. One of the characteristic features of Hartmannella lobifera n. sp. is its remarkable cyst structure.
Key Words: Amoeba; Gymnamoebia; Paramoebidae; Hartmannellidae; Ultrastructure.
Introduction The number of named species of marine amoebae is still relatively low, compared with that of freshwater and soil species (PAGE 1983a, 1991). Some workers have studied marine amoebae without species identification or detailed descriptions (PENNICK & GOODFELLOW 1975; ANDERSON 1977), and so the presence of the members of some genera in salt water is known with certainty whereas very few or no forms are described at the specific level (PAGE 1983a). Only one marine species of Hartmannella is known (PAGE 1980) and no taxonomically valid descriptions of marine species of Dactylamoeba exist. Two representatives of these genera, isolated from the brackish-water bay (salinity about 15%0) are described in the present paper. The application of the generic name Dactylamoeba KOROTNEFF, 1880 proposed by PAGE (1982, 1983a, 1983b) was found to be taxonomically incorrect (GOODKOY 1988). One reason is that the generic name Dactylamoeba is already included by the International Commission of Zoological Nomenclature in the list of generic names with the remark that it is a synonym of the generic name Mayorella (PAGE 1982). Another rea-
son is that PAGE (l983a), when he proposed the generic name Dactylamoeba, suggested that the type species of the genus - D. elongata KOROTNEFF, 1880 - possesses microsales. This could be checked only with electron microscopy, however, this species was not studied with EM. Moreover, the description of D. elongata is insufficient: it does not contain any information which allows reasonable re-isolation of this species in future. Thus, the use of the name Dactylamoeba as the name of the nominal genus is incorrect, and the name Korotnevella, which is used in this paper, has been proposed as a substitute (see GOODKOV 1988 for details).
Material and Methods The amoebae were isolated from the superficial 1-1.5 mm of sediments from Niva Bay (Sound; 15 km South of Helsing0r, Denmark). Samples of sand with patches of white sulphur bacteria were collected and inoculated in either 90 mm Petri dishes with seawater overlay (salinity about 35%0) and two wheat grains in each dish, or on C75S agar
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(PAGE 1983a) with seawater overlay. Both species were able to live on C100S agar with overlay (op. cit.), but they multiplied more slowly then on C75S agar. The amoebae were observed and photographed adhering to the glass surface. Permanent preparations, stained with iron haematoxylin and with Kernechtrot were made as described by PAGE (1988). Feulgen staining was performed as described by LEE & PAWLOWSKI (1992). For electron microscopy, amoebae were fixed as follows: (1) in a mixture of equal volumes of 4% buffered glutaraldehyde, 2% osmium tetroxide and 0.1 M phosphate buffer (pH 7.4) for 40 min; (2) in buffered 4% glutaraldehyde with sucrose (10 mg/ml) at pH 7.4 for 30 min, followed by a rinse in buffer and postfixation with 2% osmium tetroxide for 2 hours; (3) in 2% buffered osmium tetroxide (pH 7.4) for 30 min. In all cases the amoebae were washed 3 times in the buffer after fixation. After dehydration with an ethanol series specimens were embedded in Epon. Sections were stained with a saturated solution of uranyl acetate dissolved in 70% ethanol and with Reynolds lead citrate and examined with a Zeiss EM 900 electron microscope.
Results Hartmannella lobifera n. sp. Locomotive amoebae (Figs. 1-4) were always monopodial, with an anterior hyaline cap which from time to time was obliterated by intruding granules. When the amoeba starts to move the cap may be somewhat narrower than the rest of the cell. The body of the locomotive form was usually equally wide at its anterior, posterior and middle parts. Amoebae moved by steady flowing of the cytoplasm; no evidence of eruption was seen. Seventy specimens were measured on the glass surface. The length of the locomotive form varied form 28 to 42 11m (mean 35.2 11m) and the breadth from 5 to 8 11m (mean 6.8 11m). Lengthlbreadth ratio (LIB) varied between 4 and 7 (mean 5.1). The presence of 1-3 small posterior hyaline lobes was very common (Figs. 3-4). The formation of these lobes was a very characteristic process connected with the changing of the direction of movement (see below). Some specimens in locomotion had no lobes, but instead they had a broad, not very well differentiated morulate uroid (Fig. 1) and some had no differentiated uroid at all. Villous uroid or trailing uroidal filaments were never observed. The amoebae frequently changed direction of locomotion. In fact it was difficult to find a monopodial locomotive form, because most amoebae produced pseudopodia, often in two or three different directions simultaneously (Figs. 7-8,10). The amoeba can change the direction of locomotion by turning its anterior end, but more often it produces a lateral pseudopodium. This
pseudopodium becomes the leading one and the amoeba starts to move in a new direction. While the major part of the amoeba body progresses in the new direction the remnants of the previous hyaline can remain at the same place. Thus it moves along the amoeba body towards its posterior end (Figs. 5-6). When it reaches the end of the cell it separates from the glass and· forms a posterior hyaline lobe. When not moving ("resting") the amoebae were generally rounded, often with 3-5 distinct hyaline lobes (Fig. 9). The amoebae did not produce a differentiated floating form; floating amoebae were often irregular and produced pseudopodia in all directions. A vesicular nucleus was discernible in both live (Fig. 13) and in stained (Fig. 14) specimens. Its diameter as measured in stained preparations, was 3.5-6 11m and the diameter of the nucleolus was I-211m. A small contractile vacuole was usually situated in the posterior part of the body (Figs. 5-6), but sometimes it was observed moving within the cell (Fig. 2). Prior to systole it was always situated near the surface at the posterior part of the cell. The refractile spheres, about 1 11m in diameter, were the most characteristic cytoplasmic inclusions. Their number varied from 3 to 42 per cell. The cytoplasm also contained many small opaque granules and some food vacuoles. After 3-4 weeks of cultivation on medium (2) the amoebae produced cysts (Figs. 11-12). The cysts had two distinct walls: a thin outer wall and a thicker inner one. The cell was sealed to the inner wall of the cyst. The slightly ovoid endocyst was always situated eccentrically inside the spherical ectocyst, being attached to the outer wall from one side and leaving a distinct empty space on the other side. No cyst pores were detected. The diameter of the ectocyst was 10-12 11m and the maximum dimension of the endocyst was 8-11 11m. All observed cysts were uninucleate. The fine structure of this species was found to be typical of Hartmannellids. The nucleus (Figs. 15-16) was spherical, with a central and slightly heterogeneous nucleolus (Fig. 16) and without any differentiated inner nuclear lamina. The cell surface coat (Fig. 17) consisted of a thin (about 20 nm) glycocalyx without any visible differentiation. The mitochondria (Fig. 18) were rounded or elongate, with tubular cristae. Dictyosomes (Fig. 19) were few and consisted of 3-6 flattened cisternae. Some membrane-bounded inclusions (Fig. 20) with a rounded or irregular form, were present in sections. Fig. 21 shows the contractile vacuole, which was situated in the posterior part of the body, near the cell surface. It was surrounded by regularly arranged microfilaments and by small vesicles; several mitochondria were always situated around it. In all studied cells the granuloplasm was separated from the hyaloplasm by a layer of microfilaments (Fig. 22).
Hartmannella lobifera n. sp. and Korotnevella nivo n. sp.
Microfilaments were also abundant in the granuloplasm and sometimes they were arranged in short cores, but careful searching did not indicate the presence of cytoplasmic microtubules.
285
A study of the cysts (Figs. 23-26) indicated the presence of three amorphous distinct layers in the endocyst. The total thickness of the endocyst wall was about 170 nm. The ectocyst was finer and its wall appeared to
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Figs. 15-22. Hartmannella lobifera: 15 - total section of the cell; 16 - nucleus; 17 - cell surface; 18 - mitochondria; 19dictyosome; 20 - microbody-like structure; 21 - contractile vacuole; 22 - microfilaments at the border of the granuloplasm and the hyaloplasm. (Abbreviations: cv - contractile vacuole; m - mitochondria; mf - microfilaments.) Scale bars: Figs. 17 and 19 - 0.25 11m; others - 0.5 11m.
Hartmannella lobi/era n. sp. and Korotllevella Ilivo n. sp.
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Figs. 23-26. Hartmannella lobifera: 23 - total section of the cyst; 24 - cyst wall in cross section; 25 - gutters in saggital section; 26 - gutters in cross section. Figs. 27 and 28. Korotnevella nivo: 27 - nucleus; 28 - scales. (Abbreviations: c cytoplasm; eb - electron-dense body; em - patches of electron-dense material; et - electron transparent zone; g - channels; is - internal space of the cyst; iw - inner cyst wall; ow - outer cyst wall.) Scale bar 0.5 ~m throughout.
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have a filamentous nature. Its thickness was about 160 nm. The endocyst was always situated eccentrically within the ectocyst (Fig. 23) and a distinct eccentric empty space between them was always observed. Within the agar, ectocysts of neighbouring cysts may be closely adjacent one to another (Fig. 26). The surface of the cell within the cyst had channels (Figs. 25-26) which formed invaginations of the cell surface. These channels were approximately parallel at neighbouring sites on the surface, but could have different orientations in different regions of the surface (Fig. 25). The material of the inner cyst wall penetrated some of these channels (Fig. 26), while the others were empty in sections. Their functional significance remains unknown.
Korotnevella nivo n. sp. During locomotion (Figs. 29-33) the amoebae had a variable shape; usually they were elongate or more or less triangular. They produced only hyaline subpseudopodia (Figs. 29-32) which were short and finger-shaped or long (up to 1.5 times the body length) and conical. The latter type is very characteristic for this species. The cell could sometimes be broad and triangular, with 3-5 anteriorly directed, long pseudopodia. In this case it resembled some marine members of the genus Vexillifer (PAGE 1983a). The locomotive form without long pseudopodia resembled that of Neoparamoeba pemaquidensis (PAGE 1970, 1983a; as Paramoeba) or the limnic Korotnevella stella (see PAGE 1991). The frontal hyaloplasm always formed the border which had a variable width (Figs. 29-30) and which was more flattened than the granuloplasmic region of the body. The pseudopodia often continued as ridges on the dorsal surface of the amoeba (Fig. 31) and three or four of these ridges were usually present in the locomotive form. In most cases there was no differentiated uroid, but sometimes the posterior end of the amoeba was plicate or irregularly morulate (Fig. 33). During continuous locomotion the amoebae always lacked differentiated uroidal structures. Eighty-five live specimens were measured in the culture dishes with the use of a water immersion objective. The length of the locomotive form varied between 19 11m and 51 11m (mean 39 11m); the breadth varied between 14 11m and 40 11m (mean 21 11m). Mean lengthlbreadth ratio (LIB) was l.9 (range: 0.95-2.8). The subpseudopodia never determined the direction of locomotion. In order to change the direction of movement the amoeba formed a broad wave of hyaloplasm
which was thrown out in the current direction of movement. When resting the amoeba had an irregular shape (Fig. 34) or rounded (Fig. 35) with many short, hyaline finger-shaped projections. These projections were formed in all directions from both the lateral and the dorsal hyaloplasm. The floating form (Fig. 36) was radial and it had from three to fifteen long, tapering and pointed hyaline pseudopodia. The longest were 3-4 times the diameter of the central mass of cytoplasm. The nucleus was vesicular with a single nucleolus which was located centrally or slightly eccentrically. The nucleolus was visible in both living specimens (Fig. 37) and in haematoxylin-stained preparations (Fig. 38). The diameter of the nucleus, measured in living specimens, was 2.2-3.8 11m, that of the nucleolus was l.2-2.2 11m. In stained preparations the nucleus was 3.4-4.5 11m in diameter. A para some was not detected, neither in living specimens (Fig. 37) nor in stained preparations. Feulgenstained preparations (Fig. 39) showed a slightly Feulgenpositive nucleus with a small central or eccentric Feulgen-negative lacuna and no evidence of a parasome. DAPI staining (Fig. 40) showed a homogeneously fluorescent nucleus without discernible internal differentiation and no evidence of other fluorescent inclusions, except for the food vacuoles in some specimens. No contractile vacuole was observed. The cytoplasm was packed with different types of inclusions including small food vacuoles, optically opaque granules of different size and shape, but no crystals were detected. Cysts were not found in the cultures. Electron-microscopic examination showed that the cell coat of this species bore scales (Fig. 28). They were generally basked-shaped and looked very like the scales of Paramoeba eilhardi illustrated by GRELL & BENWITZ (1970). The length of each scale was 400-514 nm, the breadth 170-230 nm and the width about 200 nm. The nucleus (Fig. 27) had a very distinct structure. At its middle, or somewhat eccentric, a compact rounded mass of electron-dense material was situated measuring about 1.6 11m in diameter. It was surrounded by an area of less electron-dense material which formed an irregular, but generally rounded body in the center of the nucleus. In some specimens this body made direct contact with the nuclear envelope, but often there was a distinct space between the body and the nuclear envelope which was filled with patches of electron-dense material. A differentiation of the fibrillar component in the nucleus was not observed; there was no nuclear lamina. With respect to its dimensions and position, the dense central body
Figs. 29-40. Korotnevella nivo: 29-30 - in locomotion; 31 - showing dorsal ridges; 32 - with long anterior pseudopodia; 33 - without these pseudopodia; 34 - in non-directed movement; 35 - when not moving; 36 - floating form; 37 - nucleus in living specimen; 38 - haemotoxylin-stained preparation; 39 - Feulgen-stained preparation; 40 - DAPI-stained preparation. (n - nucleus.) Scale bar 10 11m throughout.
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seems to correspond to the Feulgen-negative lacuna (Fig. 39), while the less dense material surrounding it corresponds to the "endosome" which was visible in haematoxylin-stained preparations (Fig. 38). No paras orne was detected at the electron-microscopical level during the examination of serially-sectioned amoebae. A satisfactory fixation of the cytoplasm and of the organelles was not obtained, but it was possible to conclude that the mitochondria of this species had tubular cristae and an electron-dense matrix. The single large dictyosome was always situated near the nucleus. Rounded lipid bodies were common in the cytoplasm.
Systematic account Hartmannella lobifera n. sp.
This "limax" (PAGE 1981) amoeba, moving by steady flow of the cytoplasm, with tubular mitochondrial cristae and a vesicular nucleus can undoubtedly be classified as belonging to the family Hartmannellidae (VOLKONSKY, 1931) PAGE, 1974. Within this family it cannot be assigned to the genus Glaeseria VOLKONSKY, 1931 due to the absence of nuclear division in cysts. There is no evidence of the villous-bulbous uroid and it always has a hyaline cap during locomotion. These two important features distinguish the present species from members of the genus Saccamoeba (FRENZEL, 1892) BOVEE, 1972. Any relationship between this species and the only well-studied member of the genus Cashia PAGE, 1974 - C. limacoides - is questionable. The frontal hyaline cap is usually well pronounced in the present species, whereas in C. limacoides it "might be completely absent (frequently) or present as a small hyaline tip or crescent" (PAGE 1974, p. 169). The formation of a bulbous uroid is noted for C. limacoides, while in the species described in this paper the uroid, if present, is morulate. Often the amoeba has one or two posterior hyaline lobes or no differentiated uroidal structures at all. PAGE (1986, 1988) noted, that in C. limaco ides, mitochondria are never elongate and have helical cristae, while in the present species they are often elongate and show no evidence of helical cristae. The most appropriate generic home for the species described above, is the genus Hartmannella (ALEXIEFF, 1912) PAGE, 1974. The species is similar to the known representatives of this genus with respect to locomotive morphology, formation of the lateral pseudopodia while changing direction of movement and absence of a villous-bulbous uroid. The posterior hyaline lobes which are common for this species were also noted for H. cantabrigiensis (PAGE 1974, Fig. 43). The fine structure of the present species corresponds well to that known
from studied freshwater and marine species of Hartmannella (PAGE 1980,1986). Cup-like structures or any other discrete elements in the glycocalyx of the present species were not found, but they were possibly destroyed during fixation. In the marine H. abertawensis, differentiated elements in the glycocalyx, were also not observed with certainity (PAGE 1980). The present species differs from all known species of Hartmannella due to its locomotive morphology and the cyst structure. These features warrant a description of a new species - Hartmannella lobifera. Diagnosis: Hartmannella lobifera n. sp. Hyaline cap sometimes obliterated by the intruding granuloplasm. Posterior hyaline lobes common, sometimes a morulate-like uroid is present. Length of the locomotive form varies from 28 to 42 /lm (mean 35.2 /lm); breadth varies from 5 to 8 /lm (mean 6.8 /lm). Lengthlbreadth ratio (LIB) varies from 4 to 7 (mean 5.1). Nucleus 3.5-6 /lm in diameter, nucleolus 1-2 /lm. Contractile vacuole present when amoebae are cultivated in 20%0 seawater. Capable of living and multiplying in 35%0 seawater and does not survive in freshwater. No cytoplasmic crystals. Cysts 10-12 /lm in diameter. Endocyst always situated eccentrically, being attached to the ectocyst at one side and separated from the latter by a distinct empty space at the opposite side. Inner cyst wall consists of three amorphous layers; its thickness is about 170 nm. Outer wall appears to have a fibrous nature; its thickness is about 160 nm. Known habitat: The Sound, Niva Bay (brackish-water, salinity about 15%0), Denmark. Type slides are deposited at the British Natural History Museum (London, U.K.). Holotype: 1995:9:6:5; Paratype: 1995:9:6:6. Differential diagnosis: The present species is three times larger then the only known marine species of Hartmannella, H. abertawensis; it has a different uroid and a different cyst structure. The latter feature also differentiates it from any described freshwater member of this genus. Korotnevella nivo n. sp.
The combination of light- and electron-microscopical features of this species (finger-shaped hyaline pseudopodia ["dacty10podia"], absence of the parasome, radiate floating form, microsca1es) gives good reason to classify it as a member of the genus Korotnevella (PAGE, 1981) GOODKOV, 1988 (Gymnamoebia, Paramoebidae). Within this genus only two freshwater species - K. bulla and K. stella - have been studied in detail including the use of electron microscopy (PENNICK & GOODFELLOW 1975; PAGE 1981). Both are clearly distinct from the present species with respect to light-microscopical morphology. Possibly, electron-microscopic study of some
Hartmannella lobifera n. sp. and Korotnevella nivo n. sp.
strains of Mayorella sensu lato, which are described only from light-microscopic data (BOVEE & SAWYER 1979; PAGE 1983a, 1991 and references therein), will show them to be scale-bearing. PENNICK & GOODFELLOW (1975) illustrated (using TEM) two unidentified marine scale-bearing amoebae (designated as Mayorella sp. 1 and Mayorella sp. 2). The first species has scales which are clearly distinct from those of the present species. There are some similarities between the scales of the present species and those of "Mayorella sp. 2", but the absence of any lightmicroscopic descriptions of these species leaves no possibility for further comparison. An unidentified parasome-free amoeba strain studied by GRELL & BENWITZ (1970) has scales closely resembling those of the present species (and of P. eilhardi). But in the case of this species, the authors (op. cit.) stressed a close morphological similarity to P. eilhardi while the present species is much smaller and has different pseudopodia. An unidentified strain studied by ANDERSON (1977), is more similar to the present species in dimensions and has scales of the same shape, though smaller than those of the present species. The pattern of nucleolar organisation, illustrated by ANDERSON (1977, p. 371) for his strain is different to that described here, but the absence of a light-microscopical description of ANDERSON'S strain does not allow for a more detailed comparison. The above discussion warrants the description of a new species - Korotnevella nivo (the specific name corresponds to the Danish pronunciation of the name of the bay where the species was found). Comparison of the information given by ANDERSON (1977) and GRELL & BENWITZ (1970) with the present description suggests that at least three different species of marine Korotnevella exist (given that the strain, studied by GRELL & BENWITZ is not a parasome-free strain of P. eilhardi). They all seem to show morphological differences, but all have the same general pattern of scales. Scales of P. eilhardi also closely resemble scales of these species. In contrast, two freshwater species, K. stella and K. bulla, each has a distinct scale pattern. Recently a freshwater Korotnevella was isolated and studied (SMIRNOV, unpublished). It has discoid scales with a conical projection in the middle of each scale. Such scales are different from those of any described species, but they closely resemble those of "Mayorella sp. 3" (PENNICK & GOODFELLOW 1975, Plate 5, Fig. e). This indicates that the taxonomic importance of such features as shape and dimensions of the scales in amoebae needs further detailed study. Diagnosis: Korotnevella nivo n. sp. Body triangular or elongate during locomotion. Subpseudopodia hyaline, short finger-shaped or long conical (up to 1.5 times body length). Mean length of the
291
locomotive form 39 f.lm (19-51 f.lm), mean breadth 21 f.lm (14-40 f.lm). Mean lengthlbreadth ratio (LIB) 1.9 ranging between 0.95 and 2.8. Nuclear diameter 2.2-3.8 f.lm in live specimens and 3.4-4.5 f.lm in stained preparations. Capable of living and multiplying in seawater (35%0) and does not survive in freshwater. Length of each scale 400-514 nm, breadth 170-230 nm and width about 200 nm Known habitat: The Sound, Niva Bay (brackish-water, salinity about 15%0), Denmark. Type slides are deposited at the British Natural History Museum (London, U.K.). Holotype: 1995:2:9:3, Paratype: 1995:2.9:4. This isolate is deposited with the CCAP No. 1543/1. Differential diagnosis: Korotnevella nivo differs well from both freshwater species of this genus by the tendency to produce very long anterior hyaline pseudopodia. It does not survive in freshwater. It has a nucleus with a distinct structure which differs from that of any freshwater scale-bearing species. At the light-microscopic level it may be confused with some marine Vexillifera, but the structure of the cell coat allows it to be differentiated from the members of this genus.
Acknowledgements: This work was carried out during my stay at the Marine Biological Laboratory, University of Copenhagen, supported by a Danish Government Scholarship. I am most grateful to Prof. T. FENCHEL for providing the laboratory facilities and for helpful discussions on the paper. N. V. LENTS MAN helped me greatly during preparation of the text; Dr. A. B. GOODKOV made valuable comments to the manuscript. The work was supported with the Danish Natural Science Research Grant 11-0088-1 provided to Prof. T. FENCHEL.
References ANDERSON, O. R. (1977): Fine structure of a marine ameba associated with a blue-green alga in the Sargasso Sea. J. Protozoo!. 24: 370-376. GOODKOV, A. V. (1988): Korotnevella nom. nov. - new generic name for scale-bearing Mayorella-like amoebae. Zoo!. Zmn. 67: 1728-1730 (in Russian). GRELL, K. G. & BENWITZ, G. (1970): Ultrastruktur mariner Amoeben. I. Paramoeba eilhardi SCHAUDlNN. Arch. Protistenkd. 112: 119-137. LEE, J. J. & PAWLOWSKI, I. (1992): Feulgen staining the nuclei offoraminifera. In: LEE, 1. 1. & SOLDO, F. 1. (eds.), Protocols in Protozoology. Soc. of Protozoologists, Kansas, C l2-J-C 12-1. PAGE, F. C. (1970): Two new species of Paramoeba from Maine. 1. Protozool. 17: 421-427. - (1974): A further study of taxonomic criteria for limax amoebae, with descriptions of new species and a key to genera. Arch. Protistenkd. 116: 149-184. - (1980): A light- and electron-microscopical comparison of limax and flabellate marine amoebae belonging to four genera. Protistologica 16: 57-78.
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- (1981): Mayorella SCHAEFFER, 1926 and Hollandella n. g. (Gymnamoebia), distinguished by surface structure and other characters with comparison of three species. Protistologica 17: 543-562. - (1982): Mayorella SCHAEFFER, 1926 (Rhizopoda: Amoebida): proposed conservation. Bull. Zool. Nom. 39: 214-217. - (1983a): Marine gymnamoebae. Inst. Terr. Ecol., Cambridge. - (1983b): Three freshwater species of Mayorella (Amoebida) with a cuticle. Arch. Protistenkd. 127: 203-233. - (1986): The limax amoebae: comparative fine structure of the Hartmannellidae (Lobo sea) and further comparisons with the Vahlkampfiidae (Heterolobosea). Protisto logic a 21: 361-383. - (1988): A new key to freshwater and soil gymnamoebae. Freshwater BioI. Ass., Ambleside, U. K.
- (1991): Nackte Rhizopoda. Protozoenfauna. Bd. 2. Stuttgart, New York. PENNICK, N. C. & GOODFELLOW, L. P. (1975): Some observations on the cell surface structures of species of Mayorella and Paramoeba. Arch. Protistenkd. 117: 41-46. Accepted: August 21, 1996 Published: April 1997
Author's address: ALEXEY V. SMIRNOV, Department of Invertebrate Zoology, Faculty of Biology & Soil Sci., St. Petersburg State University, Universitetskaja nab. 7/9, 199034, St. Petersburg, Russia. FAX: +7 812 2180852; e-mail: [email protected]