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Protistology 2 (3), 178-184 (2002)
Protistology
Re-description of Vannella mira Schaeffer 1926 (Gymnamoebia, Vannellidae), an often mentioned but poorly known amoebae species
Alexey V. Smirnov
Marine Biological Laboratory University of Copenhagen, Helsingor, Denmark and Department of Invertebrate Zoology, Faculty of Biology and Soil Sci, St.Petersburg State University
Summary
Amoebae species Vannella mira Schaeffer 1926 was relatively well described and has often been discovered in various regional faunas. However, there is still no clear understanding as to what we really mean by the name “ Vannella mira”. A.A. Schaeffer described this species from marine environment in 1926 (as Flabellula mira), but further researchers applied this name for several brackish- and freshwater isolates. F.C. Page re-described “ V. mira” in 1968 from freshwater and in 1976 listed it among freshwater amoebae. Perhaps, it was a misidentification. In 1988 he revised his opinion stating that “long experience has failed to reveal any other gymnamoeba occurring in both fresh and salt water” and applied a specific name Vannella cirifera Frenzel 1892 to the isolate that he previously called V. mira. However, various authors used the name V. mira for different marine and freshwater isolates even after the re-naming. The present paper describes a marine amoeba isolated from cyanobacterial mats in France (Camarque) that corresponds in full to Schaeffer’s initial description of “Flabellula mira”. New type material is established for the species V. mira Schaeffer 1926; it remains a type species of the genus Vannella.
Key words: amoebae, Vannella, Lobosea, Gymnamoebia, morphology, systematics, Vannellidae
Introduction
An amoebae species “Flabellula mira” was established by A.A. Schaeffer (1926) with the following diagnosis: “Length in locomotion 25 |j,m; breadth 15 to 25 |j,m; shape oval to fan-shaped in outline; when not in locomotion, shape extremely variable with numerous pseudopods of a large variety of shapes. Rate of locomotion rapid, 75 |j,m per minute. Endoplasm clear in anterior portion, finely granular in posterior part. A small number of refractory grains of irregular
shape always present in endoplasm. No vacuoles. No crystals. Nucleus single. Chromatin mass spherical, about 2 |j,m in diameter. Nuclear membrane 5 |j,m in diameter. Food vacuoles in small number 2 to 5 |j,m in diameter. No uroid. Food, bacteria. Habitat, sea-water, among blue-green algae, and elsewhere; common. Tortugas and Key West, Florida.” The above diagnosis is accomplished with a rather detailed description (Schaeffer, 1926, p. 48-49) and eleven line drawings, of which six illustrates the floating form, three -transformation from floating to locomotive form, one
© 2002 by Russia, Protistology
shows a sequence of the body shape in locomotion and the last is a summarising coloured drawing of the locomotive form (op. cit., fig. 12 and plate 4, fig.1). No type material was established (it was not a rule at that time).
It seems that this relatively well-described (for the facilities of that time) species should not produce much difficulties to systematists, given that up to now species distinction within the genus Vannella (to which Schaeffer’s species belongs in the modern system of amoebae) is based on the same features that are mentioned in Schaeffer’s description. Hopkins (1938) reported that the marine amoeba which he identified as “Flabellula mira” could be cultured in 5 ppt seawater, though without forming contractile vacuole. Bovee in his PhD thesis (1950, cited by Bovee and Sawyer, 1979) mentioned this species from marine habitats, and three years later (Bovee, 1953a) isolated a strain that he called “Flabellula mira” Schaeffer 1926 from a freshwater pond and noted the presence of contractile vacuole in the cytoplasm of this strain. He argued that his strain is indistinguishable from Schaeffer’s one and believed that the contractile vacuole is developed under freshwater conditions, but is absent in marine isolate.
Actually, this mentioning introduced a misunderstanding. Given that “F. mira” may be found in both marine and freshwater, Page (1968), basing on the observations of two freshwater isolates, re-defined the species “F. mira”. He formulated a new, rather detailed diagnosis of this species (not an emendation of Schaeffer’s one) (Page, 1968, p. 13). According to Page’s definition, “F. mira”has the “greatest dimension of the locomotive form approximately 23-55 |j,m; greatest dimension usually breadth. Diameter of the nucleus 4.8—8.3 |j,m; diameter of nucleolus 2.1—4.1 |j,m” (op. cit., p. 14). His strain could grow on the agar made with up to 10 ppt seawater and one strain, abnormal in appearance - up to 25 ppt. As a distinctive feature of his strain he noted the presence of numerous faecal pellets, visible on agar surface. No type material was established.
Bovee (1965) re-defined Flabellula and established the genus Vannella for the species that did not fit to the former genus. Former “F. mira”was transferred to the newly established taxon as a type species and named Vannella mira Schaeffer 1926. Page (1969) recognised these changes and wrote that the species “F. mira” which he had re-described in 1968, should therefore be identified as Vannella mira (Schaeffer, 1926), thus stating identity of his and Schaeffer’s species. Page (1971) mentioned this species also as Vannella mira and noted that “while V. mira (Schaeffer, 1926) ... were originally described from salt water, amoebae indistinguishable in form from V. mira was found in fresh
water (e.g. Page, 1968)”. In his first key to freshwater and soil amoebae (Page, 1976), he illustrated the strain that he called V. mira with photographs (p. 101) and give the dimensions according to his data (Page, 1968). Some amoebae in his photographs bore few faecal pellets. Page (1979) gave a re-diagnosis of the genus Vannella with the same type species — Vannella mira Schaeffer 1926. Page and Blakey (1979) illustrated the cell coat of freshwater V. mira.
At the same time Bovee and Sawyer (1979) described V. mira among marine amoebae (distribution: Atlantic coast, US, Florida to Maine, Pacific coast, Southern California; coral reefs, Madagascar). The distribution mentioned far exceeds that noted by Schaeffer (1926) and suggests that Bovee and Sawyer isolated this species themselves from various marine locations. According to their description, this species in locomotion is 15 to 25 |j,m long by 15 to 25 |j,m wide, nucleus 4 to 6 |j,m in diameter with central endosome 2 to 3 |j,m in diameter. Contractile vacuole absent in seawater, 3 to 6 |j,m in diameter in brackish or fresh water. These measurements of the contractile vacuole differ from that given by Page (1968), namely 4.7—7.6 |j,m. As a source of other data they refer to Schaeffer (1926) and Page (1971). Bovee (1985) gives the same description and measurements of V. mira, but refers to Schaeffer (1926) only.
Later Page (1988) stated that “long experience has failed to reveal any other gymnamoeba occurring in both fresh and salt water” and suggested a name Vannella cirifera Frenzel 1892 as a substitute for the strain that he called V. mira in 1968. However, no other motivation except that “Frenzel’s name has priority, and his figures (Frenzel, 1892) greatly resembles those in this publication” was given (1988, p. 74). One ofthe photographs of “V. cirifera ” given by Page (1988) is the same as for “V. mira” in Page (1976); two other are different. Page (1991, p. 87) gives three new photographs of “V. cirifera”. Page (1988) illustrates the cell coat of “V. cirifera”, stating that, in contrast with V. simplex, this species does now have simple filaments among glycostyles.
Several authors (Davis et al., 1978; Fernandez et al., 1989; Munson, 1992; Fernandez-Leborans and Novillo, 1993; Rogerson and Gwaltney, 2000) list V. mira in the faunas of marine habitats. Michel and Just (1984) mentioned “ V. mira” among the species isolated from the cooling and rinsing water in dental treatment units — i.e. from freshwater. Ariza et al. (1994) illustrated two freshwater strains called V. mira.
Growing bulk of knowledge about amoebae indicated that species distinction among vannellids is very difficult (Page, 1988; Smirnov, 2001). However, there are at least 14 morphologically different species
of the genus Vannella (Page, 1991; 1983; Smirnov and Fenchel, 1996; Smirnov, 2001), corresponding to the modern (Page, 1979) definition of the genus. This number does not include V. mira or V. cirifera, because of the difficulties in the species definition mentioned above. The impression is that various authors applied the name V. mira to different species. To stop the misuse of the name of the type species of the genus Vannella, a marine amoebae strain that corresponds in full to the original Schaeffer’s description of V. mira is illustrated and described in the present paper; neotype of V. mira is established.
Material and Methods
Vannella mira was isolated from cyanobacterial mats collected in Camarque, (France, Mediterranean coast). At the time of sampling the water salinity at the sampling point was about 50 ppt, but it may undergo significant variations. A stratified multi-layered mat, consisting mostly of cyanobacteria with underlying sandy sediment, was collected, dispersed and inoculated on the site into 200 ml cell culture flasks (Greiner) containing 42 ppt. artificial seawater (Wiegandt Gmbh, Germany) and one wheat grain. After cloning (by capturing a single floating form with a micro-pipette), cultures were maintained in 60 mm Petri dishes filled with the same artificial seawater, with the addition of either one wheat grain per dish or 5 ml of1% cerophyl infusion (Page, 1988) per dish.
For electron microscopical examination, amoebae were fixed by adding several drops of 2% osmium tetroxide to a 10 day-old culture. Prior to fixation, the overlying water was decanted off so that approximately 1 mm layer of culture media remain on the bottom of the dish. After 1-2 minutes, the medium in the dish was replaced with 0.5% osmium tetroxide in 0.1M phosphate buffer (pH 7.4). Amoebae were fixed for 30 minutes, washed three times in phosphate buffer (pH 7.4), dehydrated through an ethanol series and embedded in Taab 812 resin.
Results and Discussion
1. Vannella mira: description
Typical locomotive form of Camarque isolate (Figs
1—5; 14, A—E) was fan-shaped (Fig. 14, A, B, E) sometimes with pronounced “tail” (Fig. 14, C), semicircular or (rarely) spatulate (Fig. 14, D). Frontal hyaloplasm constituted up to half of the body, usually it also formed anterior hyaline crescent. Length in locomotion, measured in culture on the plastic surface was 15—35 |j,m (average 28.5 |J.m), breadth 15—35 |j,m
(average 26.5 |j,m). Length/breadth ratio 0.5—1.8 (average 1.08). In a few specimens there were small dorso-lateral ridges or small lobes at the frontal hyaloplasm (Figs 2; 5; 14, D), but most ofcell in culture had smooth surface. Some amoebae in culture bore several rounded faecal pellets. Developed floating form (Figs 6—9; 14, F—L) always had long, narrow-conical pseudopodia with rounded tips; their number varied from three to eleven. Several very characteristic types of the floating form (Fig. 14, F—L) were observed:
1. Floating form with 3—11 radiating pseudopodia; some pseudopodia may furcate. During the development of the floating form pseudopodia elongate, but sometimes the development may “freeze” and floating amoeba for a long time has short, curved, relatively thick pseudopodia (e.g. Fig. 14, F).
2. Floating form with all cytoplasm in the conical basal thickenings of 3—4 pseudopodia (in this case the basement of pseudopodia consists of the granuloplasm) (e.g. Fig. 14, H).
3. Floating form with 7—11 pseudopodia, producing the impression of the cell rotating, with pseudopodia lagging behind (e.g. Fig.14, I).
The single nucleus was of vesicular type (Fig. 10), about 6 |j,m in diameter, with the central rounded nucleolus, about 3 |jm in diameter. No crystals. Cysts were never observed. In most of the cells there was a large vacuole, resembling contractile, but it never contracted in 42 ppt water.
TEM studies revealed routine ultrastructure (Figs
11—13), typical for all vannellians. Mitochondria had tubular cristae; well pronounced dictyosomes consisted of 3—6 cisternae. No simple filaments were found among glycostyles; the last were about 130 nm in length.
2. Systematic account
It is often difficult to motivate species distinction among vannellians at the morphological level, and perhaps molecular or other non-morphological characters must be applied in future for species description and comparison (Sims et al., 1999; Smirnov et al., 2002). Anyway, it would be a wrong approach to reject all older species descriptions, especially those containing characteristic features that may help in identification, and to rename all species. Camarque isolate of Vannella show very characteristic features that allow to relate it with Schaefer’s description of “Flabellula mira”.
Among characters that are not common for all vannellians and can be used for comparison of species are the size of locomotive form, organisation and dimensions of the nucleus and, especially, floating form. Schaeffer (1926) left a very detailed and well-organised
Figs 1-13. Light- and electron-microscopic photographs of Vannella mira. 1-5 - locomotive forms; 69 - floating forms; 10 - nucleus (arrowhead); 11 - nucleus; 12 - mitochondria and dictyosome; 13 -cell coat. Scale bars: 1-10 - 10 |im, 11-13 - 500 nm.
description of these features. Summarising his description and drawings, “Flabellula mira” is an amoeba 15—25 |jm in length and breadth (thus, usual length/ breadth ration should be about 1.0; it is confirmed with his drawings), semicircular, spatulate or fan-shaped, with occasional unevenness on the frontal hyaloplasm. It has
vesicular nucleus, about 5 |j,m in diameter and relatively small nucleolus, about 2 |j,m in diameter. Characteristic appearances of the floating form are:
— Central mass with several irregularly curved or coiled pseudopodia (Schaeffer, 1926, p. 48, fig. 12 (4)).
Fig. 14. Vannella mira (schematic drawings). A-E: typical appearances of the locomotive form. F-L: floating forms (not to scale).
— Central mass with several straight, regularly arranged pseudopodia, producing the appearance of a cell rotating, with pseudopodia lagging behind (op. cit., p. 48, fig. 12 (5)).
— Floating form with nearly all cytoplasm in several pseudopodia. Each pseudopodium has a distinct conical basal thickening (op. cit., p. 48, fig. 12 (7)).
Perhaps, the organisation of the floating form, rather stable and characteristic for amoebae (Schaeffer, 1926; Bovee, 1953b; Page, 1976, 1988) is ofthe primary importance in case ofvannellians, the second one being the dimentions and nuclear structure. From this point of view, the description of “Flabellula mira” (Page, 1968) does not correspond to Schaeffer’s description in the organisation of the floating form (none of the indicated characteristic patterns is illustrated or described by Page) and size (locomotive form ofPage’s strain is nearly two times larger; the same is true for the nucleus). In other words, it does not fit the diagnosis and description of “Flabellulamira”. Page’s motivation of the identification of this strain “resemble F. mira Schaeffer more than they resemble any other described species, also they are somewhat smaller than Schaeffer’s strain” cannot be considered satisfactory. There is something strange with the measurements — Page’s strain is larger, not smaller than Schaeffer’s. Page gave re-diagnosis of“F. mira”. We have to conclude that F.C. Page (1968) misidentified his strain, and incorrectly applied the name “Flabellula mira” (in future Vannella
mira). Thus, we consider Page’s re-diagnosis of the species “Flabellula mira” (Page, 1968) incorrect due to the misidentification ofdescribed strain. Page’s strain had to be described as another species at this time.
Later Page (1988) suggested a new identification for the strain that he previously called “ Vannella mira”
— Vannella cirifera Frenzel 1892, initially described by Frenzel (1892) as Saccamoeba cirifera. However, Frenzel (1892) left a very briefdescription ofhis species, without any characteristic details. Page (1988) did not argue the change of species name in any details other than the general similarity and freshwater habitat of V. cirifera (in contrast with marine V. mira). Description of V. cirifera given by Page (without re-diagnosis) resembles V. simplex but not marine V. mira. Page & Blakey (1979) illustrate cell coat of “V. mira”, Page (1988) — that of “V. cirifera”. There is no clear indication if it is the TEM of the same strain (photographs are different), but we know that the culture named V. mira was kept in the Culture Collection of Algae and Protozoa (UK) for some time (Page and Blakey (1979) indicated the reference number). This strain was lost and is no more listed in CCAP.
The only seemingly clear distinctive feature — absence of simple filaments in V. cirifera (Page, 1988) is shown to be non-reliable during the morphological and molecular study of various strains of Vannella simplex (Smirnov et al., 2002). Presence on numerous phaecal pellets in culture of V. mira, noted by Page (1968, as Flabellula) and respectively expanded to V. cirifera (Page, 1988) also is shown to be a feature of V. simplex (Smirnov et al., 2002). Thus, we have to conclude that the description of species Vannella cirifera contains no distinctive features allowing identification and the name V. cirifera (Frenzel 1892) Page 1988 must be announced “nomen dubium”. Perhaps, F.C. Page maintained and studied a strain of a large freshwater Vannella, different from both marine V. mira Schaeffer 1926 and freshwater V. simplex Wohlfarth-Bottermann 1960, but unfortunately this strain was lost and no type material that may allow establishing neotype of V. cirifera remains.
The present description of Camarque strain of Vannella corresponds to Schaeffer’s description of Vannella mira in all three mentioned characteristic details of the appearance of floating form and in both locomotive form (the last is only slightly larger in average measurements) and nucleus sizes (also only slightly larger). Mentioned size differences are deep within the range of interspecific polymorphism, known for many amoebae species (e.g. see Smirnov, 1999). Camarque isolate corresponds to the description of V. mira by Sawyer and Bovee (1979) and Bovee (1985). Additionally, this strain was isolated from the same type
of habitat as Schaeffer’s isolate (blue-green algae). It warrants the announcement of the studied strain as Vannella mira (Schaeffer, 1926). To leave no formal taxonomic problems, re-diagnosis of V. mira and new type material are given below. Vannella mira so far remains among marine amoebae species and remains a type species of the genus Vannella Bovee 1965.
Diagnosis
Vannella mira Schaeffer 1926, emend.
In locomotion fan-shaped, semicircular or (rarely) spatulate. Prominent folds or lobes on the frontal hyaloplasm. Length and breadth of the locomotive form 15—35 |j,m; (average length 28.5 |j,m; average breadth 26.5 |j,m) l/b ratio 0.5—1.8 (average 1.08). Single vesicular nucleus, about 6 |j,m in diameter with central spherical endosome about 3 |j,m in diameter. Floating form non-symmetrical, with up to 11 straight or coiled pseudopodia, often with distinct conical thickening of the basement. Pseudopodia of the floating form have rounded, not pointed ends and are relatively thick.
Food: bacteriovorous. Habitat: marine, brackish water. Ecotopes: cyanobacterial mats, known from shallow coastal waters, bays, tide pools, coral reefs. Distribution: Atlantic and Pacific US coats, Florida, Madagascar, Camarque (France, Mediterranean coast).
Type material: neotype, culture (clone) Vannella mira CCAP 1589/15.
Differential diagnosis: similar in size with V. septentrionalis, V. arabica, V. caledonica and V. anglica. Differs from all these species in the organisation of the floating from. In addition, V. arabica has distinct simple filaments among glycostyles, that were not found in V. mira (this feature may be not very reliable). V. anglica has a characteristic uneven frontal edge, that is not a feature of V. mira (see Page, 1979).
Acknowledgements
The work in Marine Biological Laboratory was financed by the European Commission (contract no. EVK3-CT-1999-00010 MATBIOPOL) and the Danish Natural Science Research Council (contract no. 9700549); further studies with the state program “Russian Universities” No. 992619 and INTAS YSF 2001/2-0048 grant.
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Address for correspondence: A.V. Smirnov, Dept. of Invertebrate Zoology, Faculty of Biology and Soil Sci., St. Petersburg State University, 199034, Universitetskaja nab. 7/9, St. Petersburg, Russia.
E-mail: smirnov@home.rclph.spbu.ru
The manuscript is presented by A.V.Goodkov
