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 shape always present in endoplasm. No vacuoles. No crystals. Nucleus single. Chromatin mass spherical, An amoebae species “Flabellula mira” was esta about 2 µm in diameter. Nuclear membrane 5 µm in blished by A.A. Schaeffer (1926) with the following diameter. Food vacuoles in small number 2 to 5 µm in diagnosis: “Length in locomotion 25 µm; breadth 15 diameter. No uroid. Food, bacteria. Habitat, sea water, to 25 µm; shape oval to fan shaped in outline; when among blue green algae, and elsewhere; common. not in locomotion, shape extremely variable with Tortugas and Key West, Florida.” The above diagnosis numerous pseudopods of a large variety of shapes. Rate is accomplished with a rather detailed description of locomotion rapid, 75 µm per minute. Endoplasm (Schaeffer, 1926, p. 48–49) and eleven line drawings, clear in anterior portion, finely granular in posterior of which six illustrates the floating form, three – part. A small number of refractory grains of irregular transformation from floating to locomotive form, one
© 2002 by Russia, Protistology Protistology · 179 shows a sequence of the body shape in locomotion and water (e.g. Page, 1968)”. In his first key to freshwater the last is a summarising coloured drawing of the and soil amoebae (Page, 1976), he illustrated the strain locomotive form (op. cit., fig. 12 and plate 4, fig.1). that he called V. mira with photographs (p. 101) and No type material was established (it was not a rule at give the dimensions according to his data (Page, 1968). that time). Some amoebae in his photographs bore few faecal It seems that this relatively well described (for the pellets. Page (1979) gave a re diagnosis of the genus facilities of that time) species should not produce much Vannella with the same type species – Vannella mira difficulties to systematists, given that up to now species Schaeffer 1926. Page and Blakey (1979) illustrated the distinction within the genus Vannella (to which cell coat of freshwater V. mira. Schaeffer’s species belongs in the modern system of At the same time Bovee and Sawyer (1979) amoebae) is based on the same features that are described V. mira among marine amoebae (distribution: mentioned in Schaeffer’s description. Hopkins (1938) Atlantic coast, US, Florida to Maine, Pacific coast, reported that the marine amoeba which he identified Southern California; coral reefs, Madagascar). The as “Flabellula mira” could be cultured in 5 ppt seawater, distribution mentioned far exceeds that noted by though without forming contractile vacuole. Bovee in Schaeffer (1926) and suggests that Bovee and Sawyer his PhD thesis (1950, cited by Bovee and Sawyer, 1979) isolated this species themselves from various marine mentioned this species from marine habitats, and three locations. According to their description, this species years later (Bovee, 1953a) isolated a strain that he called in locomotion is 15 to 25 µm long by 15 to 25 µm wide, “Flabellula mira” Schaeffer 1926 from a freshwater nucleus 4 to 6 µm in diameter with central endosome 2 pond and noted the presence of contractile vacuole in to 3 µm in diameter. Contractile vacuole absent in the cytoplasm of this strain. He argued that his strain is seawater, 3 to 6 µm in diameter in brackish or fresh indistinguishable from Schaeffer’s one and believed that water. These measurements of the contractile vacuole the contractile vacuole is developed under freshwater differ from that given by Page (1968), namely 4.7–7.6 conditions, but is absent in marine isolate. µm. As a source of other data they refer to Schaeffer Actually, this mentioning introduced a misun (1926) and Page (1971). Bovee (1985) gives the same derstanding. Given that “F. mira” may be found in both description and measurements of V. mira, but refers to marine and freshwater, Page (1968), basing on the Schaeffer (1926) only. observations of two freshwater isolates, re defined the Later Page (1988) stated that “long experience has species “F. mira”. He formulated a new, rather detailed failed to reveal any other gymnamoeba occurring in diagnosis of this species (not an emendation of both fresh and salt water” and suggested a name Schaeffer’s one) (Page, 1968, p. 13). According to Vannella cirifera Frenzel 1892 as a substitute for the Page’s definition, “F. mira” has the “greatest dimension strain that he called V. mira in 1968. However, no other of the locomotive form approximately 23–55 µm; motivation except that “Frenzel’s name has priority, greatest dimension usually breadth. Diameter of the and his figures (Frenzel, 1892) greatly resembles those nucleus 4.8–8.3 µm; diameter of nucleolus 2.1–4.1 in this publication” was given (1988, p. 74). One of the µm” (op. cit., p. 14). His strain could grow on the agar photographs of “V. cirifera” given by Page (1988) is the made with up to 10 ppt seawater and one strain, same as for “V. mira” in Page (1976); two other are abnormal in appearance – up to 25 ppt. As a distinctive different. Page (1991, p. 87) gives three new photo feature of his strain he noted the presence of numerous graphs of “V. cirifera”. Page (1988) illustrates the cell faecal pellets, visible on agar surface. No type material coat of “V. cirifera”, stating that, in contrast with V. was established. simplex, this species does now have simple filaments Bovee (1965) re defined Flabellula and established among glycostyles. the genus Vannella for the species that did not fit to the Several authors (Davis et al., 1978; Fernandez et former genus. Former “F. mira” was transferred to the al., 1989; Munson, 1992; Fernandez Leborans and newly established taxon as a type species and named Novillo, 1993; Rogerson and Gwaltney, 2000) list V. Vannella mira Schaeffer 1926. Page (1969) recognised mira in the faunas of marine habitats. Michel and Just these changes and wrote that the species “F. mira” (1984) mentioned “V. mira” among the species isolated which he had re described in 1968, should therefore from the cooling and rinsing water in dental treatment be identified as Vannella mira (Schaeffer, 1926), thus units – i.e. from freshwater. Ariza et al. (1994) stating identity of his and Schaeffer’s species. Page illustrated two freshwater strains called V. mira. (1971) mentioned this species also as Vannella mira and Growing bulk of knowledge about amoebae noted that “while V. mira (Schaeffer, 1926) … were indicated that species distinction among vannellids is originally described from salt water, amoebae indis very difficult (Page, 1988; Smirnov, 2001). However, tinguishable in form from V. mira was found in fresh there are at least 14 morphologically different species 180 · Alexey V. Smirnov of the genus Vannella (Page, 1991; 1983; Smirnov and (average 26.5 µm). Length/breadth ratio 0.5–1.8 Fenchel, 1996; Smirnov, 2001), corresponding to the (average 1.08). In a few specimens there were small modern (Page, 1979) definition of the genus. This dorso lateral ridges or small lobes at the frontal number does not include V. mira or V. cirifera, because hyaloplasm (Figs 2; 5; 14, D), but most of cell in culture of the difficulties in the species definition mentioned had smooth surface. Some amoebae in culture bore above. The impression is that various authors applied several rounded faecal pellets. Developed floating form the name V. mira to different species. To stop the misuse (Figs 6–9; 14, F–L) always had long, narrow conical of the name of the type species of the genus Vannella, a pseudopodia with rounded tips; their number varied marine amoebae strain that corresponds in full to the from three to eleven. Several very characteristic types original Schaeffer’s description of V. mira is illustrated of the floating form (Fig. 14, F–L) were observed: and described in the present paper; neotype of V. mira 1. Floating form with 3–11 radiating pseudopodia; is established. some pseudopodia may furcate. During the develop ment of the floating form pseudopodia elongate, but Material and Methods sometimes the development may “freeze” and floating amoeba for a long time has short, curved, relatively Vannella mira was isolated from cyanobacterial thick pseudopodia (e.g. Fig. 14, F). mats collected in Camarque, (France, Mediterranean 2. Floating form with all cytoplasm in the conical coast). At the time of sampling the water salinity at the basal thickenings of 3–4 pseudopodia (in this case the sampling point was about 50 ppt, but it may undergo basement of pseudopodia consists of the granuloplasm) significant variations. A stratified multi layered mat, (e.g. Fig. 14, H). consisting mostly of cyanobacteria with underlying 3. Floating form with 7–11 pseudopodia, produ sandy sediment, was collected, dispersed and inoculated cing the impression of the cell rotating, with pseudo on the site into 200 ml cell culture flasks (Greiner) podia lagging behind (e.g. Fig.14, I). containing 42 ppt. artificial seawater (Wiegandt Gmbh, The single nucleus was of vesicular type (Fig. 10), Germany) and one wheat grain. After cloning (by about 6 µm in diameter, with the central rounded capturing a single floating form with a micro pipette), nucleolus, about 3 µm in diameter. No crystals. Cysts cultures were maintained in 60 mm Petri dishes filled were never observed. In most of the cells there was a with the same artificial seawater, with the addition of large vacuole, resembling contractile, but it never either one wheat grain per dish or 5 ml of 1% cerophyl contracted in 42 ppt water. infusion (Page, 1988) per dish. TEM studies revealed routine ultrastructure (Figs For electron microscopical examination, amoebae 11–13), typical for all vannellians. Mitochondria had were fixed by adding several drops of 2% osmium tubular cristae; well pronounced dictyosomes consisted tetroxide to a 10 day old culture. Prior to fixation, the of 3–6 cisternae. No simple filaments were found overlying water was decanted off so that approximately among glycostyles; the last were about 130 nm in length. 1 mm layer of culture media remain on the bottom of the dish. After 1 2 minutes, the medium in the dish 2. SYSTEMATIC ACCOUNT was replaced with 0.5% osmium tetroxide in 0.1M phosphate buffer (pH 7.4). Amoebae were fixed for 30 It is often difficult to motivate species distinction minutes, washed three times in phosphate buffer (pH among vannellians at the morphological level, and 7.4), dehydrated through an ethanol series and perhaps molecular or other non morphological embedded in Taab 812 resin. characters must be applied in future for species description and comparison (Sims et al., 1999; Smirnov Results and Discussion 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 1. VANNELLA MIRA: DESCRIPTION identification, and to rename all species. Camarque Typical locomotive form of Camarque isolate (Figs isolate of Vannella show very characteristic features that 1–5; 14, A–E) was fan shaped (Fig. 14, A, B, E) allow to relate it with Schaefer’s description of sometimes with pronounced “tail” (Fig. 14, C), “Flabellula mira”. semicircular or (rarely) spatulate (Fig. 14, D). Frontal Among characters that are not common for all hyaloplasm constituted up to half of the body, usually vannellians and can be used for comparison of species it also formed anterior hyaline crescent. Length in are the size of locomotive form, organisation and locomotion, measured in culture on the plastic surface dimensions of the nucleus and, especially, floating form. was 15–35 µm (average 28.5 µm), breadth 15–35 µm Schaeffer (1926) left a very detailed and well organised Protistology · 181
Figs 1 13. Light and electron microscopic photographs of Vannella mira. 1 5 locomotive forms; 6 9 floating forms; 10 nucleus (arrowhead); 11 nucleus; 12 mitochondria and dictyosome; 13 cell coat. Scale bars: 1 10 10 µm, 11 13 500 nm.
description of these features. Summarising his des vesicular nucleus, about 5 µm in diameter and relatively cription and drawings, “Flabellula mira” is an amoeba small nucleolus, about 2 µm in diameter. Characteristic 15–25 µm in length and breadth (thus, usual length/ appearances of the floating form are: breadth ration should be about 1.0; it is confirmed with his drawings), semicircular, spatulate or fan shaped, with – Central mass with several irregularly curved or occasional unevenness on the frontal hyaloplasm. It has coiled pseudopodia (Schaeffer, 1926, p. 48, fig. 12 (4)). 182 · Alexey V. Smirnov
mira). Thus, we consider Page’s re diagnosis of the species “Flabellula mira” (Page, 1968) incorrect due to the misidentification of described 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 brief description of his 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 indi cation if it is the TEM of the same strain (photographs Fig. 14. Vannella mira (schematic are different), but we know that the culture named V. drawings). A E: typical appearances mira was kept in the Culture Collection of Algae and of the locomotive form. F L: floating Protozoa (UK) for some time (Page and Blakey (1979) forms (not to scale). indicated the reference number). This strain was lost and is no more listed in CCAP. The only seemingly clear distinctive feature – – Central mass with several straight, regularly absence of simple filaments in V. cirifera (Page, 1988) arranged pseudopodia, producing the appearance of a is shown to be non reliable during the morphological cell rotating, with pseudopodia lagging behind (op. cit., and molecular study of various strains of Vannella p. 48, fig. 12 (5)). simplex (Smirnov et al., 2002). Presence on numerous – Floating form with nearly all cytoplasm in phaecal pellets in culture of V. mira, noted by Page several pseudopodia. Each pseudopodium has a distinct (1968, as Flabellula) and respectively expanded to V. conical basal thickening (op. cit., p. 48, fig. 12 (7)). cirifera (Page, 1988) also is shown to be a feature of V. simplex (Smirnov et al., 2002). Thus, we have to Perhaps, the organisation of the floating form, conclude that the description of species Vannella cirifera rather stable and characteristic for amoebae (Schaeffer, contains no distinctive features allowing identification 1926; Bovee, 1953b; Page, 1976, 1988) is of the primary and the name V. cirifera (Frenzel 1892) Page 1988 must importance in case of vannellians, the second one being be announced “nomen dubium”. Perhaps, F.C. Page the dimentions and nuclear structure. From this point maintained and studied a strain of a large freshwater of view, the description of “Flabellula mira” (Page, Vannella, different from both marine V. mira Schaeffer 1968) does not correspond to Schaeffer’s description 1926 and freshwater V. simplex Wohlfarth Bottermann in the organisation of the floating form (none of the 1960, but unfortunately this strain was lost and no type indicated characteristic patterns is illustrated or material that may allow establishing neotype of V. described by Page) and size (locomotive form of Page’s cirifera remains. strain is nearly two times larger; the same is true for the The present description of Camarque strain of nucleus). In other words, it does not fit the diagnosis Vannella corresponds to Schaeffer’s description of and description of “Flabellula mira”. Page’s motivation Vannella mira in all three mentioned characteristic of the identification of this strain “resemble F. mira details of the appearance of floating form and in both Schaeffer more than they resemble any other described locomotive form (the last is only slightly larger in species, also they are somewhat smaller than Schaeffer’s average measurements) and nucleus sizes (also only strain” cannot be considered satisfactory. There is slightly larger). Mentioned size differences are deep something strange with the measurements – Page’s within the range of interspecific polymorphism, known strain is larger, not smaller than Schaeffer’s. Page gave for many amoebae species (e.g. see Smirnov, 1999). re diagnosis of “F. mira”. We have to conclude that F.C. Camarque isolate corresponds to the description of V. Page (1968) misidentified his strain, and incorrectly mira by Sawyer and Bovee (1979) and Bovee (1985). applied the name “Flabellula mira” (in future Vannella Additionally, this strain was isolated from the same type Protistology · 183 of habitat as Schaeffer’s isolate (blue green algae). It Vannella isolated from freshwater. Microbiologia. warrants the announcement of the studied strain as 5, 25–33. Vannella mira (Schaeffer, 1926). To leave no formal Bovee E.C. 1950. Some observations based on the use taxonomic problems, re diagnosis of V. mira and new of phase contrast microscope, concerning the type material are given below. Vannella mira so far taxonomy and morphology of certain free living remains among marine amoebae species and remains amoebas. Ph.D. Thesis, Univ. Calif., Los Angeles. a type species of the genus Vannella Bovee 1965. Bovee E.C. 1953a. Presence of the contractile vacuole in Flabellula mira Schaeffer in fresh water. Proc. Diagnosis Soc. Protozool. 4, 15. Bovee E.C. 1953b. Morphological identification of free living amoebida. Proc. Iowa Acad. Sci. 60, VANNELLA MIRA SCHAEFFER 1926, EMEND. 599–615. In locomotion fan shaped, semicircular or (rarely) Bovee E.C. 1965. An emendation of the amoeba genus spatulate. Prominent folds or lobes on the frontal Flabellula and a description of Vannella gen. nov. hyaloplasm. Length and breadth of the locomotive form Trans. Amer. Microsc. Soc. 84, 217–227. 15–35 µm; (average length 28.5 µm; average breadth Bovee E.C. and Sawyer T.K. 1979. Marine flora and 26.5 µm) l/b ratio 0.5–1.8 (average 1.08). Single fauna of the North Western United States. vesicular nucleus, about 6 µm in diameter with central Protozoa. Sarcodina: Amoebae. N.O.A.A. Techn. spherical endosome about 3 µm in diameter. Floating Rep. NMFS, Circ. 419, 1–56. form non symmetrical, with up to 11 straight or coiled Bovee E.C. 1985. Class Lobosea Carpenter, 1861. In: pseudopodia, often with distinct conical thickening of An Illustrated Guide to the Protozoa (Eds. Lee J.J., the basement. Pseudopodia of the floating form have Hutner S.H. and Bovee E.C.). Allen Press, rounded, not pointed ends and are relatively thick. Lawrence. pp. 158–211 Food: bacteriovorous. Habitat: marine, brackish Davis P.G., Caron D.A. and Sieburth J.McN. 1978. water. Ecotopes: cyanobacterial mats, known from Oceanic amoebae from the north atlantic: culture, shallow coastal waters, bays, tide pools, coral reefs. distribution, and taxonomy. Trans. Amer. Micros. Distribution: Atlantic and Pacific US coats, Florida, Soc. 97, 73–88. Madagascar, Camarque (France, Mediterranean coast). Fernandes M C. A., Crespo E.P., Mallen M.M., Ares Type material: neotype, culture (clone) Vannella M.P. and Casas M.L.C. 1989. Marine amoebae mira CCAP 1589/15. from waters of Northwest Spain, with comments on a potentially pathogenic euryhaline species. J. 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Description of four species of the genus Protozool. 15, 9–26. 184 · Alexey V. Smirnov
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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: [email protected]
The manuscript is presented by A.V.Goodkov